Methods for determining protein structure using a surface-selective nonlinear optical technique

ABSTRACT

Methods, devices, and systems are disclosed for determining protein structure and dynamics using second harmonic generation (SHG) and related surface-selective nonlinear optical techniques.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/142,314, filed Apr. 2, 2015, and also claims the benefit of U.S.Provisional Application No. 62/148,649, filed Apr. 16, 2015, both ofwhich applications are incorporated herein by reference in theirentirety.

BACKGROUND

Second harmonic generation (SHG) is a nonlinear optical process whichmay be configured as a surface-selective detection technique thatenables detection of binding interactions and conformational change inproteins and other biological targets using second harmonic-activelabels attached to the target molecules (see, for example, U.S. Pat.Nos. 6,953,694, and 8,497,073). To date these methods have been appliedto detect ligand-induced conformational changes in a variety of systemsand to distinguish ligands by the type of conformation they induce uponbinding (Salafsky, J. S. (2001), “‘SHG-labels’ for Detection ofMolecules by Second Harmonic Generation”, Chemical Physics Letters 342,485-491; Salafsky, J. S. (2003), “Second-Harmonic Generation as a Probeof Conformational Change in Molecules”, Chemical Physics Letters 381,705-709; Salafsky, J. S. (2006), “Detection of Protein ConformationalChange by Optical Second-Harmonic Generation”, Journal of ChemicalPhysics 125; Moree, B., et al. (2015), “Small Molecules Detected bySecond Harmonic Generation Modulate the Conformation of Monomericα-Synuclein and Reuce Its Aggregation in Cells”, J. boil. Chem. 290(46);27582-27593; Moree, et al. (2015), “Protein Conformational Changes areDetected and Resolved Site Specifically by Second-Harmonic Generation”,Biophys. J. 109:806-815). Examples include distinguishing between type Ivs. type II kinase inhibitors, such as imatinib and dasatinib, whichbind to the protein to induce inactive and active conformations,respectively.

SHG and the related technique sum-frequency generation (SFG) have beenused in the past to study the orientation of dye molecules at aninterface (Heinz T., et al., (1983), “Determination of MolecularOrientation of Monolayer Adsorbates by Optical Second-HarmonicGeneration”, Physical Review A 28(3):1883-1885; Heinz, T, (1991)Second-Order Nonlinear Optical Effects at Surfaces and Interfaces”, inNonlinear Surface Electromagnetic Phenomena (Stegeman, H. P. a. G. ed.),Elsevier, Amsterdam, pp 353-416). In these measurements, the componentsof the nonlinear susceptibility (χ⁽²⁾) of the labeled interface aredetermined using polarized light. Details of the molecular orientationdistribution for the dye molecules at the interface can then be inferredusing the experimentally determined values for χ⁽²⁾ and assumptionsregarding the degree of orientation of the dye molecules within theplane of the interface, the relative magnitude of the components ofhyperpolarizability (α⁽²⁾) of the dye molecules in the molecular frameof reference, etc.

The field of protein and biomolecular structure determination is highlydeveloped but there remains a need for a sensitive and rapid measure ofconformational change and structure in real time and in solution. Mostinformation about protein structure and dynamics has come mainly fromX-ray crystallography and NMR studies, but these techniques arerelatively labor and material intensive, slow, or provide only a staticsnapshot of protein structure.

The presently disclosed methods, devices, and systems for determiningprotein structure using surface-selective nonlinear optical techniquesaddress these unmet needs. In some embodiments, determination of proteinstructure in a high-throughput format is enabled through the use ofnovel device designs and mechanisms for rapid, precise, andinterchangeable positioning of substrates (comprising the tethered orimmobilized biological targets to be analyzed) with respect to theoptical system used to deliver excitation light, and which at the sametime ensure that efficient optical coupling between the excitation lightand the substrate surface is maintained. One preferred format forhigh-throughput optical interrogation of biological samples is theglass-bottomed microwell plate. The systems and methods disclosed hereinprovide mechanisms for coupling the high intensity excitation lightrequired for SHG and other nonlinear optical techniques to a substrate,e.g. the glass substrate in a glass-bottomed microwell plate, by meansof total internal reflection in a manner that is compatible with therequirements for a high-throughput analysis system.

SUMMARY

Disclosed herein are methods for determining protein structure insolution, the methods comprising: (a) tethering protein molecules to asurface under a first set of experimental conditions, wherein theprotein molecules are labeled at one or more known positions with one ormore nonlinear-active labels; (b) illuminating the tethered proteinmolecules of step (a) with excitation light of at least one fundamentalfrequency, wherein the excitation light is provided by at least onelight source; (c) detecting a first physical property of light generatedby the one or more nonlinear-active labels as a result of theillumination in step (b); (d) tethering the protein molecules on asurface under at least a second set of experimental conditions; (e)illuminating the tethered protein molecules of step (d) with excitationlight of at least one fundamental frequency; (f) detecting at least asecond physical property of light generated by the one or morenonlinear-active labels as a result of the illumination in step (e); and(g) comparing the at least second physical property of the lightdetected in step (f) to the first physical property of the lightdetected in step (c) to determine a relative orientation of the one ormore nonlinear-active labels in the tethered protein molecules.

In some embodiments, the methods further comprising globally fittingdata for the relative orientation of the one or more nonlinear-activelabels to a structural model of the protein molecule, wherein thestructural model is based on known positions of the one or morenonlinear-active labels within the protein molecule.

In some embodiments, the methods further comprise repeating steps (a)through (f) for at least two different nonlinear-active label-proteinconjugates, wherein the nonlinear-active labels are attached to at leasttwo different sites on the protein molecule. In some embodiments, the atleast two different nonlinear-active label-protein conjugates eachcomprise a single-site cysteine. In some embodiments, thenonlinear-active labels are nonlinear-active unnatural amino acids. Insome embodiments, the at least second physical property of light isdifferent from the first physical property of light. In someembodiments, the first and the at least second physical properties oflight possess the same polarization but are of different magnitudes orintensities. In some embodiments, the first and at the least secondphysical properties of light possess different polarizations. In someembodiments, the nonlinear-active unnatural amino acid is Aladan or aderivative of naphthalene. In some embodiments, the methods furthercomprise incorporating x-ray crystallographic data for the protein intothe structural model of the protein molecule. In some embodiments, theprotein molecules are labeled at two or more known positions. In someembodiments, the protein molecules are labeled at three or more knownpositions.

In some embodiments, steps (d) through (f) are repeated for at least athird set of experimental conditions. In some embodiments, steps (d)through (f) are repeated for at least a fourth set of experimentalconditions. In some embodiments, the first set of experimentalconditions comprises applying a first electric field of a first electricfield strength to the tethered protein molecules, and the at leastsecond set of experimental conditions comprises applying an at leastsecond electric field of an at least a second electric field strength tothe tethered protein molecules. In some embodiments, the first electricfield and the at least second electric field are direct current (DC)fields. In some embodiments, the first electric field and the at leastsecond electric field are alternating current (AC) fields. In someembodiments, the first electric field is a direct current (DC) field andthe at least second electric field is an alternating current (AC) field.In some embodiments, the first electric field is an alternating current(AC) field and the at least second electric field is a direct current(DC) field. In some embodiments, the first electric field and the atleast second electric field are applied using an array of electrodesfabricated on the surface. In some embodiments, the array of electrodesis a circular array as illustrated in FIG. 9.

In some embodiments, the first set of experimental conditions comprisestethering the protein molecules using a His-tag attached to theN-terminus, and the at least second set of experimental conditionscomprises tethering the protein molecules using a His-tag attached tothe C-terminus. In some embodiments, the first set of experimentalconditions comprises tethering the protein molecules using a firstHis-tag selected from the group consisting of 2×His, 4×His, 6×His,8×His, 10×His, 12×His, and 14×His, and the at least second set ofexperimental conditions comprises tethering the protein molecules usingan at least second His-tag that differs in length from the firstHis-tag.

In some embodiments, the first set of experimental conditions comprisestethering the protein molecules using a first assay buffer, and the atleast second set of experimental conditions comprises tethering theprotein molecules using an at least second assay buffer that differsfrom the first assay buffer. In some embodiments, the difference betweenthe first assay buffer and the at least second assay buffer is selectedfrom the group consisting of ionic strength, pH, detergentconcentration, calcium ion (Ca2+) concentration, magnesium ion (Mg2+)concentration, polyethylene glycol concentration, and any combinationthereof.

In some embodiments, the difference between the first set ofexperimental conditions and the at least second set of experimentalconditions comprises contacting the tethered protein molecules with atleast a first ligand that is known to bind to and induce conformationalchange in the protein molecules.

In some embodiments, the one or more nonlinear-active labels located atthe one or more known positions are the same. In some embodiments, theone or more nonlinear-active labels located at the one or more knownpositions are different. In some embodiments, the one or morenonlinear-active labels are second harmonic (SH)-active labels. In someembodiments, the one or more nonlinear-active labels are sum frequency(SF)-active labels. In some embodiments, the one or more nonlinearactive labels are difference frequency (DF)-active labels.

In some embodiments, the illuminating steps comprise adjusting thepolarization of excitation light of at least one fundamental frequency.In some embodiments, the detecting in steps (c) and (e) compriseadjusting the polarization of the light generated by the one or morenonlinear-active labels that reaches a detector. In some embodiments,the first and at least second physical properties of light are intensityor polarization.

Also disclosed herein are methods for determining the absoluteorientation of a nonlinear-active label attached to a tethered protein,the method comprising: (a) detecting a physical property of lightgenerated by a nonlinear-active surface as a result of illumination withexcitation light of at least one fundamental frequency, whereindetection is performed using two different polarization states of theexcitation light; (b) detecting a physical property of light generatedby a nonlinear-active labeled protein tethered in an oriented fashion ona non-labeled surface, wherein the light is generated as a result ofillumination with excitation light of the at least one fundamentalfrequency, and wherein detection is performed using two differentpolarization states of the excitation light; (c) detecting a physicalproperty of light generated by a nonlinear-active labeled proteintethered in an oriented fashion on a nonlinear-active labeled surface,wherein the light is generated as a result of illumination withexcitation light of the at least one fundamental frequency, and whereindetection is performed using the two different polarization states ofthe excitation light; and (d) determining the absolute orientation ofthe nonlinear-active label attached to the tethered protein by comparingthe physical property of the light in step (a), the physical property ofthe light detected in step (b), and the physical property of the lightdetected in step (c).

In some embodiments, the excitation light is directed to the surface insuch a way that it is totally internally reflected from the surface. Insome embodiments, a first polarization state of the excitation lightcomprises p-polarization relative to its plane of incidence, and asecond polarization state of the excitation light comprisess-polarization relative to its plane of incidence. In some embodiments,the methods further comprise detecting a physical property of the lightgenerated in steps (a), (b), and (c) using excitation light of more thantwo different polarization states.

In some embodiments, the nonlinear-active labeled surface is preparedusing covalent carbodiimide coupling of a carboxylated nonlinear-activelabel to an aminosilane-functionalized glass substrate surface. In someembodiments, the nonlinear-active labeled surface comprises a supportedlipid bilayer, and wherein the supported lipid bilayer further comprisesan amine- or thiol-containing lipid to which a nonlinear-active label iscovalently coupled.

In some embodiments, the nonlinear-active labeled protein is tethered inan oriented fashion on the non-labeled or nonlinear-active labeledsurface using covalent carbodiimide coupling of the C-terminus of theprotein to an aminosilane-functionalized glass substrate surface. Insome embodiments, the nonlinear-active labeled protein is tethered in anoriented fashion on a non-labeled or nonlinear-active labeled surfacecomprising a supported lipid bilayer, and wherein the nonlinear-activelabeled protein is inserted into the supported lipid bilayer or attachedto an anchor molecule that is inserted into the supported lipid bilayer.

In some embodiments, the nonlinear-active label is a second harmonic(SH)-active label. In some embodiments, the nonlinear-active label is asum frequency (SF)-active label. In some embodiments, thenonlinear-active label is a difference frequency (DF)-active label.

Disclosed herein are devices comprising: (a) a substrate comprising afirst surface that further comprises a plurality of discrete regions,wherein each discrete region further comprises a supported lipid bilayerand a patterned array of electrodes; and (b) a well-forming componentbonded to or integrated with the first surface of the substrate so thateach discrete region is contained within a single well.

In some embodiments, a plurality of supported lipid bilayers furthercomprises a nonlinear-active labeled protein. In some embodiments, theplurality of supported lipid bilayers further comprises anonlinear-active protein that is the same for each. In some embodiments,the plurality of supported lipid bilayers further comprise two or moresubsets of supported lipid bilayers, and wherein each subset ofsupported lipid bilayers comprises a different nonlinear-active protein.In some embodiments, the substrate is fabricated from anoptically-transparent material selected from the group consisting ofglass, fused-silica, polymer, or any combination thereof. In someembodiments, the patterned array of electrodes comprises an array of twoor more electrodes patterned on the substrate surface surrounding thesupported lipid bilayer. In some embodiments, the patterned array ofelectrodes comprises an array of two or more electrodes patterned on thewalls of each well of the well-forming unit. In some embodiments, thepatterned array of electrodes comprises at least one electrode patternedon a lid that seals each well. In some embodiments, the well-formingunit comprises 96 wells. In some embodiments, the well-forming unitcomprises 384 wells. In some embodiments, the well-forming unitcomprises 1,536 wells. In some embodiments, the device further comprisesan array of prisms integrated with a second surface of the substrate andconfigured to deliver excitation light to the first surface of thesubstrate so that it is totally internally reflected from the firstsurface.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. In the event of a conflictbetween a term herein and a term in an incorporated reference, the termherein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A provides a schematic illustration of the energy level diagramsfor fluorescence (an absorption/emission process).

FIG. 1B provides a schematic illustration of the energy level diagramsfor second harmonic generation (a two photon scattering process).

FIG. 2 provides a schematic illustration of a conformational change in aprotein (labeled with a nonlinear-active moiety) which is induced bybinding of a ligand, and its impact on the orientation of anonlinear-active label relative to an optical interface (Z-axis) towhich the protein is attached.

FIG. 3 illustrates the relationship between the laboratory frame ofreference (as defined by X, Y, and Z axes) and the molecular frame ofreference (as defined by X′, Y′, and Z′ axes). For some nonlinear-activemolecules, the hyperpolarizability tensor (α⁽²⁾) may be dominated by asingle component in the molecular frame of reference, i.e., α⁽²⁾=α⁽²⁾_(Z′Z′Z′).

FIG. 4 illustrates one non-limiting example of the system architecturefor a high throughput analysis system for determining structure orconformational change of biological molecules, e.g. proteins or otherbiological entities, based on nonlinear optical detection.

FIG. 5 shows a schematic for one non-limiting example of an opticalsetup used for analysis of structure or conformational change inbiological molecules using nonlinear optical detection.

FIG. 6 shows a schematic illustration depicting the use of a prism todirect excitation light at an appropriate incident angle such that theexcitation light undergoes total internal reflection at the top surfaceof a substrate. The two dashed lines to the right of the prism indicatethe optical path of the reflected excitation light and the nonlinearoptical signal generated at the substrate surface when nonlinear-activespecies are tethered to the surface. The substrate is optionallyconnected to the actuator of an X-Y translation stage for re-positioningbetween measurements. The curved lines between the top surface of theprism and the lower surface of the substrate indicate the presence athin layer (not to scale) of index-matching fluid used to ensure highoptical coupling efficiency between the prism and substrate.

FIG. 7 shows a schematic illustration depicting the use of a layer ofindex-matching elastomeric material attached or adjacent to the lowersurface of a transparent substrate (configured in a microwell plateformat in this example) to ensure high optical coupling efficiencybetween a prism and the upper surface of the substrate. In someembodiments of this approach, the upper surface of the prism is slightlydomed to focus the compression force when bringing the microwell plateand prism into contact, thereby reducing or eliminating the formation ofair gaps between the prism and elastomeric material.

FIG. 8 provides a schematic illustration of one non-limiting example ofa device for performing high-throughput structure determination usingsurface-selective nonlinear-optical techniques, wherein an array ofhemispherical prisms bonded to or integrated with the substrate in aglass-bottom microplate format are used to provide good optical couplingof the excitation light to the top surface of the substrate.

FIG. 9 provides a schematic illustration of one non-limiting example ofa device comprising a patterned array of electrodes surrounding an areaof a substrate surface used to form a supported lipid bilayer.

FIGS. 10A-B illustrate a microwell plate with integrated prism array forproviding good optical coupling of the excitation light to the topsurface of the substrate. Such a device may be useful in conductinghigh-throughput structure determination of proteins and other biologicalmolecules. FIG. 10A: top axonometric view. FIG. 10B: bottom axonometricview.

FIGS. 11A-B show exploded views of the microwell plate device shown inFIGS. 10A-B. FIG. 11A: top axonometric view. FIG. 11B: bottomaxonometric view.

FIG. 12 illustrates the incident and exit light paths for coupling theexcitation light to the substrate surface via total internal reflectionusing the design concept illustrated in FIGS. 10A-B.

FIGS. 13A-B show examples of data for the SHG signal intensitiesobserved for cysteine-labeled mutants of a protein molecule(dihydrofolate reductase (DHFR)) tethered to an optical interface. FIG.13A: Data for cysteine-labeled DHFR mutants tethered to a Ni/NTA-dopedsupported lipid bilayer by means of an N-terminal His-tag. FIG. 13B:Data for cysteine-labeled DHFR mutants tethered to a Ni/NTA-dopedsupported lipid bilayer by means of a C-terminal His-tag.

FIGS. 14A-B show examples of data for the percent change in SHG signalintensities observed for cysteine-labeled mutants of a protein molecule(dihydrofolate reductase (DHFR)) tethered to an optical interfacefollowing the addition of a known ligand (trimethoprim (TMP)). FIG. 14A:Data for cysteine-labeled DHFR mutants tethered to a Ni/NTA-dopedsupported lipid bilayer by means of an N-terminal His-tag. FIG. 14B:Data for cysteine-labeled DHFR mutants tethered to a Ni/NTA-dopedsupported lipid bilayer by means of a C-terminal His-tag.

FIG. 15 illustrates a computer system that may be configured to controlthe operation of the systems disclosed herein.

FIG. 16 is a block diagram illustrating a first example architecture ofa computer system that can be used in connection with exampleembodiments of the present invention.

FIG. 17 is a diagram showing one embodiment of a network with aplurality of computer systems, a plurality of cell phones and personaldata assistants, and Network Attached Storage (NAS).

FIG. 18 is a block diagram of a multiprocessor computer system using ashared virtual address memory space in accordance with an exampleembodiment.

DETAILED DESCRIPTION

The systems and methods disclosed herein relate to the field ofbiomolecular structure and dynamics determination. Methods fordetermining the relative and/or absolute orientation of secondharmonic-active labels (or other nonlinear-active labels, e.g. sumfrequency-active or difference frequency-active labels) attached toproteins or other biological molecules, and for determining molecularstructures therefrom are described. In addition, devices and systems aredescribed which are suitable for high throughput analysis of molecularorientation or molecular structure. In some aspects of the presentdisclosure, methods and systems are described for determiningorientation, conformation, structure, or changes in orientation,conformation, or structure of biological entities in response tocontacting the biological entities with one or more test entities. Asused herein, determining orientation, conformation, structure, orchanges thereof may involve measurement of a nonlinear optical signalwhich is related to and/or proportional to the average orientation of anonlinear-active label or tag. As used herein, “high throughput” refersto the ability to perform rapid analysis of molecular orientation,conformation, structure, or changes thereof for a plurality ofbiological entities optionally contacted with one or more test entities,or to the ability to perform rapid analysis of molecular orientation,conformation, structure, or changes thereof for one or more biologicalentities optionally contacted with a large plurality of test entities,or to any combination of these modalities.

In general, the methods, devices, and systems disclosed rely on the useof second harmonic generation (SHG), or the related nonlinear opticaltechniques of sum frequency generation (SHG) or difference frequencygeneration (DFG), for the determination of molecular orientation,conformation, structure, or changes thereof. In these methods,polarization-dependent measurements are used to determine the componentsof the nonlinear susceptibility, χ⁽²⁾, of protein molecules (or otherbiological molecules) oriented at a surface or interface and labeled atone or more specific sites by a hyperpolarizable moiety according tomethods known to those of skill in the art. The components of χ⁽²⁾ inturn are related to the molecular orientational distribution at eachlabel site through a model. By measuring at least two differentcomponents of χ⁽²⁾ under two or more different sets of experimentalconditions (defined below) using labels placed at one or more differentsites in the protein, one can use the resulting information on relativeorientation of the labels to develop a model of protein structure anddetect changes thereof using SHG or related nonlinear opticaltechniques.

Detection of Orientation, Conformation, and Structure Using SecondHarmonic Generation

Second harmonic generation, in contrast to the more widely usedfluorescence-based techniques (FIG. 1A), is a nonlinear optical processin which two photons of the same excitation wavelength or frequencyinteract with a nonlinear material and are re-emitted as a single photonhaving twice the energy, i.e. twice the frequency and half thewavelength, of the excitation photons (FIG. 1B). Second harmonicgeneration only occurs in nonlinear materials lacking inversion symmetry(i.e. in non-centrosymmetric materials), and requires a high intensityexcitation light source. It is a special case of sum frequencygeneration, and is related to other nonlinear optical phenomena such asdifference frequency generation. Throughout this disclosure, the termsSHG, SFG, and DFG are used interchangeably, as will be understood bythose of skill in the art.

Second harmonic generation and other nonlinear optical techniques can beconfigured as surface-selective detection techniques because of theirdependence on the orientation of the nonlinear-active species. Tetheringof the nonlinear-active species to a surface, for example, can create anet, average degree of orientation that is absent when molecules areable to undergo free diffusion in solution. An equation commonly used tomodel the orientation-dependence of nonlinear-active species at aninterface is:χ⁽²⁾ =N _(s)<α⁽²⁾>where χ⁽²⁾ is the nonlinear susceptibility, N_(s) is the total number ofnonlinear-active molecules per unit area at the interface, and <α⁽²⁾> isthe average orientation of the nonlinear hyperpolarizability (α⁽²⁾) ofthese molecules. The intensity of SHG is proportional to the square ofthe nonlinear susceptibility, and is thus dependent on both the numberof oriented nonlinear-active species at the interface and theirorientational distribution; changes in this orientational distribution,whether spatial or temporal, change the SHG intensity.

Second harmonic generation and other nonlinear optical techniques may berendered additionally surface selective through the use of totalinternal reflection as the mode for delivery of the excitation light tothe optical interface (or surface) on which nonlinear-active specieshave been tethered or immobilized. Total internal reflection of theincident excitation light creates an “evanescent wave” at the interface,which may be used to selectively excite only nonlinear-active labelsthat are in close proximity to the surface, i.e. within the spatialdecay distance of the evanescent wave, which is typically on the orderof tens of nanometers. Total internal reflection may also be used toexcite fluorescence in a surface-selective manner, for example to excitea fluorescence donor attached to the optical interface, which thentransfers energy to a suitable acceptor molecule via a fluorescenceresonance energy transfer (FRET) mechanism. In the present disclosure,the evanescent wave generated by means of total internal reflection ofthe excitation light is preferentially used to excite a nonlinear-activelabel or molecule. The efficiency of exciting nonlinear active speciesin the nonlinear-active processes described herein depends strongly ontheir average orientation relative to the surface. For example, if nonet average orientation of the nonlinear active species exists, therewill be no SHG signal.

This surface selective property of SHG and other nonlinear opticaltechniques (e.g. sum frequency generation and difference frequencygeneration) can be exploited to detect orientation, conformation,structure, and change thereof in biological molecules tethered atinterfaces. For example, conformational change in a receptor moleculedue to binding of a ligand, might be detected using a nonlinear-activelabel or moiety wherein the label is attached to or associated with thereceptor such that the conformational change leads to a change in theorientation with respect to the interface (FIG. 2), and thus to a changein a physical property of the nonlinear optical signal (e.g. a change insignal intensity or polarization). In the past, the use ofsurface-selective nonlinear optical techniques has been confined mainlyto applications in physics and chemistry, since relatively fewbiological samples are intrinsically non-linearly active. Recently, theuse of second harmonic active labels (“SHG labels”) has been introduced,allowing virtually any molecule or particle to be rendered highlynon-linear active. The first example of this was demonstrated bylabeling the protein cytochrome c with an oxazole dye and detecting theprotein conjugate at an air-water interface with second harmonicgeneration (Salafsky, J., “‘SHG-labels’ for Detection of Molecules bySecond Harmonic Generation”, Chem. Phys. Lett. 342(5-6):485-491 (2001)).

Surface-selective nonlinear optical techniques are also coherenttechniques, meaning that the fundamental and nonlinear optical lightbeams have wavefronts that propagate through space with well-definedspatial and phase relationships. The use of surface-selective nonlinearoptical detection techniques for analysis of structure and conformationof biological molecules or other biological entities has a number ofinherent advantages over other optical approaches, including: i)sensitive and direct dependence of the nonlinear signal on theorientation of the nonlinear-active species, thereby conferringsensitivity to orientation and conformational change; (ii) highersignal-to-noise (lower background) than fluorescence-based detectionsince the nonlinear optical signal is generated only at surfaces thatcreate a non-centrosymmetric system, i.e. the technique inherently has avery narrow “depth-of-field”; (iii) as a result of the narrow “depth offield”, the technique is useful when measurements must be performed inthe presence of a overlaying solution containing free (i.e., nottethered to the surface) nonlinear-active species, e.g. where a bindingprocess might be obviated or disturbed by a separation or rinse step.This aspect of the technique may be particularly useful for performingequilibrium binding measurements, which require the presence of bulkspecies, or kinetics measurements where the measurements are made over adefined period of time; (iv) the technique exhibits lowerphoto-bleaching and heating effects than those that occur influorescence, due to the fact that the two-photon absorptioncross-section is typically much lower than the one-photon absorptioncross-section for a given molecule, and that SHG (and sum frequencygeneration or difference frequency generation) involves scattering, notabsorption; (v) minimal collection optics are required and higher signalto noise is expected since the fundamental and nonlinear optical beams(e.g., second harmonic light) have well-defined incoming and outgoingdirections with respect to the interface; this is particularlyadvantageous compared to fluorescence-based detection, as fluorescenceemission is isotropic and there may also be a large fluorescencebackground component to detected signals arising from out-of-focal planefluorescent species; (vi) the signals arising from SHG, SFG or DFGprovide an instantaneous, real-time means of studying a molecule'sstructure, conformation or change thereof such as occurs, for example,upon ligand binding. This property may be very useful in the disclosedmethods for obtaining real-time “movies” of proteins undergoingstructural changes as part of their function in real time.

Determination of Protein Structure & Dynamics

Polarization Measurements:

The components of χ⁽²⁾ may be measured in the laboratory frame ofreference using polarized excitation light of at least one fundamentalfrequency. Some light sources, e.g. some lasers, produce light of afundamental frequency that is substantially polarized. In someembodiments, the polarization of the excitation light may be furtherdefined and/or adjusted using one or more optical polarizers, waveplates, etc. Typically, the plane of incidence of the polarized light(i.e. the plane defined by the propagation direction of the excitationlight and a vector perpendicular to the plane of the substrate orreflecting surface) will be the X-Z plane of the laboratory coordinatesystem illustrated in FIG. 3. Polarized light having its electric fieldvector parallel to the plane of incidence is called p-polarized light.Polarized light having its electric field vector perpendicular to theplane of incidence is called s-polarized light. In some embodiments, thepolarization of the detected second harmonic light generated byexcitation of a nonlinear-active moiety may also be defined and/oradjusted using one or more optical polarizers, wave plates, etc. Asoutlined above, by measuring at least two different components of χ⁽²⁾under two or more different sets of experimental conditions using labelsplaced at one or more different sites in the protein tethered orimmobilized in an oriented fashion on the optical interface (i.e. thesurface plane in FIG. 3), one can use the resulting information onrelative orientation of the labels to develop a model for proteinstructure and detect changes thereof using SHG or related nonlinearoptical techniques.

Proteins Labeled with Nonlinear-Active Moieties:

Attachment of nonlinear-active labels to proteins may be accomplished byany of a variety of techniques, as is well known to those of skill inthe art. Specific non-limiting examples of suitable label attachmenttechniques will be described in more detail below.

In some embodiments, one or more nonlinear-active labels may be attachedto one or more different positions within the same individual proteinmolecule. In some embodiments, the one or more nonlinear-active labelsmay be attached to one or more different positions (e.g. sites) indifferent molecules of the same protein, i.e. to create a family ofproteins comprising different versions of the labeled protein. In someembodiments, the number of labeling sites at which the protein (orfamily of proteins) is labeled may be at least 1 site, at least 2 sites,at least 3 sites, at least 4 sites, at least 5 sites, at least 6 sites,at least 7 sites, at least 8 sites, at least 9 sites, at least 10 sites,or more. In other embodiments, the nonlinear-active label may beattached to different single-site cysteine mutants or variants of thesame protein, or nonlinear-active unnatural amino acids (e.g., Aladan orother naphthalene derivatives) may be attached to a family of mutants orvariants at one or more sites. Such proteins can be engineered,naturally occurring, made using in vitro translation methods, expressedin vivo, and in general created through any of the various methods knownto those skilled in the art.

In some embodiments, the SHG measurements may comprise using proteinmolecules labeled with a single nonlinear-active label. In someembodiments, the SHG measurements may comprise using protein moleculeslabeled with at least 2 different nonlinear-active labels, at least 3different nonlinear-active labels, at least 4 different nonlinear-activelabels, at least 5 different nonlinear-active labels, at least 6different nonlinear-active labels, at least 7 different nonlinear-activelabels, at least 8 different nonlinear-active labels, at least 9different nonlinear-active labels, or at least 10 differentnonlinear-active labels.

Experimental Conditions:

As defined herein, “experimental conditions” refer to any set ofexperimental parameters under which SHG or other nonlinear opticalsignals are measured, wherein a change in one or more of theexperimental parameters in the set of experimental conditions results ina change in the measured values of χ⁽²⁾ due to a change in theunderlying molecular orientational distribution. In other words,different sets of experimental conditions produce different baseline SHGsignal intensities, different polarization dependences, differentresponses to the same ligand binding event, or any or all of theaforementioned. For example, applying an electric field to proteinsattached to a supported lipid bilayer can change the molecules'underlying orientational distribution and thus may change the measuredvalues of χ⁽²⁾. Other examples of experimental parameters that may beused to define sets of experimental conditions include, but are notlimited to, buffer conditions such as pH, ionic strength, detergentcontent and concentration, tether attachment site (e.g., through the useof N- or C-terminal His tags), etc. Specifically, the independent valuesof χ⁽²⁾ measured under the different and independent sets ofexperimental conditions permit one to obtain the underlying molecularorientational distribution in the laboratory frame of reference (FIG.3). By relating these different laboratory frame measurements to eachother, one can determine the relative difference in angle between thenonlinear-active labels positioned at two or more different label sitesin the protein frame of reference, along with other parameters of theorientational distribution such as the width of a gaussian distributionused to model the molecular orientational distribution.

In some embodiments, the use of one or more compositions of surfaces towhich the labeled proteins are tethered may be used to define differentsets of experimental conditions. For example, if supported lipidbilayers are used to tether and orient the labeled proteins, two or moredifferent lipid compositions of the bilayer (for example, with differentelectrostatic charge densities or different molar lipid dopingdensities) can be used to create two or more different orientationaldistributions of the same protein. For example, lipids with head groupsbearing different net charges can be used (e.g., zwitterionic,positively, and negatively charged lipid head groups). In someembodiments, it may be advantageous to vary the lipid composition of thesupported lipid bilayer by varying, e.g., the number of different lipidcomponents and/or their relative concentrations. Examples of lipidmolecules that may be used to form supported lipid bilayers or that maybe inserted as major or minor components of the supported lipid bilayerinclude, but are not limited to, diacylglycerol, phosphatidic acid (PA),phosphatidylethanolamine (PE), phosphatidylcholine (PC),phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositolphosphate (PIP), phosphatidylinositol biphosphate (PIP2),phosphatidylinositol triphosphate (PIP3), ceramide phosphorylcholine(sphingomyelin; SPH), ceramide phosphorylethanolamine (sphingomyelin;Cer-PE), ceramide phosphoryllipid, or any combination thereof. In someembodiments, a lipid molecule comprising a nickel-nitrilotriacetic acidchelate (Ni-NTA) moiety may be used for the purpose of tetheringproteins by means of a His tag. For example, the bilayer may incorporate1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiaceticacid)succinyl] (nickel salt) at various molar concentrations.

In some embodiments, the number of different lipid components of thelipid bilayer may range from 1 to 10, or more. In some embodiments, thenumber of different lipid components may be at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10. In some embodiments, the number of differentlipid components may be at most 10, at most 9, at most 8, at most 7, atmost 6, at most 5, at most 4, at most 3, at most 2, or at most 1.

In some embodiments, the relative percentage of a given lipid componentof the lipid bilayer may range from about 0.1% to about 100%. In someembodiments, the relative percentage of a given lipid component may beat least about 0.1%, at least about 0.2%, at least about 0.3%, at leastabout 0.4%, at least about 0.5%, at least about 1%, at least about 5%,at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, or at least about 100%. In someembodiments, the relative percentage of a given lipid component may beat most about 100%, at most about 90%, at most about 80%, at most about70%, at most about 60%, at most about 50%, at most about 40%, at mostabout 30%, at most about 20%, at most about 10%, at most about 5%, atmost about 1%, at most about 0.5%, at most about 0.4%, at most about0.3%, at most about 0.2%, or at most about 0.1%. Those of skill in theart will recognize that the relative percentage of a given lipidcomponent in the lipid bilayer may have any value within this range,e.g. about 12.5%.

If tags such as His tags are used to tether the protein, differentlengths of the His-tags (i.e., 6×, 8×, 10×, 12×, etc.) can producedifferent orientational distributions, and therefore may be used todefine different sets of experimental conditions. Moreover, N-terminalvs. C-terminal His-tags generally produce different orientationaldistributions, and thus may be used to define different sets ofexperimental conditions that yield different measured values of χ⁽²⁾.

In some embodiments, the length of the His tag used to tether a labeledprotein to a supported lipid bilayer comprising a lipid having a Ni-NTAmoiety attached may range from about 1 His residue to about 20 Hisresidues, or more. In some embodiments, the length of the His tag may beat least 1 His residue, at least 2 His residues, at least 3 Hisresidues, at least 4 His residues, at least 5 His residues, at least 6His residues, at least 7 His residues, at least 8 His residues, at least9 His residues, at least 10 His residues, at least 11 His residues, atleast 12 His residues, at least 13 His residues, at least 14 Hisresidues, at least 15 His residues, at least 16 His residues, at least17 His residues, at least 18 His residues, at least 19 His residues, orat least 20 His residues. In some embodiments, the length of the His tagmay be at most 20 His residues, at most 19 His residues, at most 18 Hisresidues, at most 17 His residues, at most 16 His residues, at most 15His residues, at most 14 His residues, at most 13 His residues, at most12 His residues, at most 11 His residues, at most 10 His residues, atmost 9 His residues, at most 8 His residues, at most 7 His residues, atmost 6 His residues, at most 5 His residues, at most 4 His residues, atmost 3 His residues, at most 2 His residues, or at most 1 His residue.

Furthermore, different buffer conditions, such as different saltconcentrations in the same buffer or different buffers, can also changethe orientational distributions of the molecules and thus the measuredvalues of χ⁽²⁾. In some embodiments, the ionic strength of the buffersused to define different sets of experimental conditions may compriseusing monovalent salts (e.g. NaCl, KCl, etc.), divalent salts (e.g.CaCl₂, MgCl₂, etc.), trivalent salts (e.g. AlCl₃), or any combinationthereof. In some embodiments, the ionic strength of the buffers used todefine different sets of experimental conditions may range from about0.0 M to about 1 M, or higher. In some embodiments, the ionic strengthof the buffer may be at least 0.0 M, at least 0.1 M, at least 0.2 M, atleast 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least0.7 M, at least 0.8 M, at least 0.9 M, or at least 1.0 M. In someembodiments, the ionic strength of the buffer may be at most 1.0 M, atmost 0.9 M, at most 0.8 M, at most 0.7 M, at most 0.6 M, at most 0.5 M,at most 0.4 M, at most 0.3 M, at most 0.2 M, or at most 0.1 M. Those ofskill in the art will recognize that the ionic strength of the buffermay have any value within this range, for example, about 0.15 M.

In some embodiments, buffer additives that associate with theinterfacial region and produce different orientational distributions ofthe proteins as a function of their concentration can also be used, suchas PEG400, ethylene glycol, etc.

In some embodiments, the number of different sets of experimentalconditions used for SHG polarization measurements may be increased inorder to increase the number of independent molecular orientationaldistributions to be sampled and the number of independent polarizationmeasurements that may be made, thereby increasing the accuracy of theangular measurements and the protein structural models derivedtherefrom. In some embodiments, the number of different sets ofexperimental conditions used may be at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 15, at least 20, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, at least 90, or at least 100.

Measurement of χ⁽²⁾ and the Relationship to Protein Structure:

Each set of experimental conditions that leads to a different set ofmeasured values for χ⁽²⁾ due to a different underlying orientationaldistribution allows for independent measurements of angle θ to bedetermined by SHG. By combining two or more such measurements, a moreaccurate determination of protein structure(s) can be made, includingstructure(s) of protein that exist in an equilibrium of multipleconformational states. Measurements of the components of χ⁽²⁾ anddetermination of the values for θ can be used to develop structuralmodels through the use of standard molecular modeling techniques knownto those of skill in the art and, in some embodiments, a choice ofappropriate simplifying assumptions. One non-limiting example of anassumption that may be made to simplify the analysis and develop proteinstructural models is that, although the orientation of the two or morelabels at different sites on the protein surface varies from oneexperimental condition to another in the laboratory frame of reference(i.e., relative to the axis normal to the surface plane), the relativeorientation between them, i.e. the angle within the protein frame ofreference remains constant under different experimental conditions. Ineffect, under this assumption one varies the orientational distributionof the proteins on the surface in ways that do not perturb theirfunction and conformational landscape. Each experimental conditionproduces at least one independent equation relating the measured SHGintensity at the different polarizations to the molecular orientationaldistribution. Appropriate controls such as ligand-induced conformationalchanges, ligand competition experiments, kinetics of ligand binding,dose-response measurements, and others, can be run at each experimentalcondition to ensure that the protein is still functional and thusnative-like. The measurements of mean angle, for example, along withother parameters of the orientational distribution of the label orhyperpolarizable moiety in the protein, can be used as constraints in denovo or integrative structural model building according to methods knownto those skilled in the art. In some embodiments, for example, an apoX-ray crystallographic structure of a protein may be included in themodel, and overlaid with structural data provided by SHG measurements toimprove the accuracy of the model.

Non-limiting examples of assumptions that may be made in someembodiments of the disclosed method for the purpose of simplifying theanalysis of the SHG structural data include: (i) that a single componentof α⁽²⁾ (e.g., α_(zzz) ⁽²⁾) dominates the hyperpolarizability of thelabel; (ii) that the hyperpolarizability of the label does not changewith changes in experimental conditions; (iii) that the position of thetwo or more labels within the protein (i.e. the identities of the aminoacid residues to which they are attached) is known; (iv) that theorientation of the tethered or immobilized protein molecules isisotropic in the X-Y plane (i.e. they are randomly oriented on the planeof the substrate surface or in the plane of a supported lipid bilayer);(v) that the change in orientation of the two or more labels is not dueto intrinsic changes in protein conformation or protein un-folding; (vi)that the orientational distribution of the label at each label siteconstitutes a delta function (i.e. that there is no spread inorientation angle at the label site), or any such combination thereof.In some embodiments, the SHG structural data may be overlaid or combinedwith structural data from protein crystallographic studies, NMR studies,UV-Vis and fluorescence spectroscopic studies, circular dichroismstudies, cross-linking experiments, small-angle X-ray scatteringstudies, etc.

In some embodiments, the attachment of nonlinear-active labels toprotein molecules may be performed using standard covalent conjugationchemistries, e.g. using non-linear active moieties that are reactivewith amine groups, carboxyl groups, thiol groups, and the like. In someembodiments, it may optionally be desirable to perform massspectrometric analysis of the labeled proteins to rigorously identifythe positions of the labeled amino acid residues within the protein.

In preferred embodiments, the attachment sites for the labels aredetermined through genetic engineering and site-directed mutagenesistechniques, as are well known to those of skill in the art (see, forexample, Edelheit, et al. (2009), “Simple and Efficient Site-DirectedMutagenesis Using Two Single-Primer Reactions in Parallel to GenerateMutants for Protein Structure-Function Studies”, BMC Biotechnology9:61). Using this approach, amino acid residues comprising amine orthiol groups, for example, may be placed at precise positions within theprotein prior to labeling with a nonlinear-active tag. Mutated proteinsmay then be tested for native-like functionality using any of a varietyof assays known to those of skill in the art, e.g, performing bindingassays using a known ligand for the protein.

In additionally preferred embodiments, genetic engineering techniquesmay be used to incorporate nonlinear-active unnatural amino acids atspecific sites within the protein using any of a variety of techniquesknown to those of skill in the art. See, for example, Cohen, et al.(2002), “Probing Protein Electrostatics with a Synthetic FuorescenceAmino Acid”, Science 296:1700-1703, and U.S. Pat. No. 9,182,406.

In one example of the disclosed methods, a protein is labeled at asingle site-specifically engineered cysteine residue with an SHG-activelabel possessing a hyperpolarizability, which in turn possesses a singledominant element of the response α⁽²⁾=α⁽²⁾ _(z′z′z′). The labeledprotein is attached via a His tag to a supported lipid bilayer membranewhich comprises Ni-NTA moieties attached to lipid headgroups. A baselineSHG signal is generated in this way, and the non-vanishing components ofχ⁽²⁾ (Salafsky, J. S. (2001), “‘SHG-labels’ for Detection of Moleculesby Second Harmonic Generation”, Chemical Physics Letters 342, 485-491;Salafsky, J. S. (2003), “Second-Harmonic Generation as a Probe ofConformational Change in Molecules”, Chemical Physics Letters 381,705-709; Salafsky, J. S. (2006), “Detection of Protein ConformationalChange by Optical Second-Harmonic Generation”, Journal of ChemicalPhysics 125) are given, as is well known to those of skill in the art:

$\begin{matrix}{{\chi_{ZZZ}^{(2)} = {N_{S}\left\langle {\cos^{3}\theta} \right\rangle\alpha_{Z\;{\prime Z}\;\prime\; Z\;\prime}^{(2)}}}{\chi_{ZXX}^{(2)} = {\chi_{XZX}^{(2)} = {\frac{1}{2}N_{S}\left\langle {\sin^{2}\theta\;\cos\;\theta} \right\rangle\alpha_{Z\;\prime\; Z\;\prime\; Z\;\prime}^{(2)}}}}} & (1)\end{matrix}$

where Ns and α_(Z′Z′Z′) ⁽²⁾ are the surface density and molecularhyperpolarizability, respectively. The components of χ⁽²⁾ can then bedetermined from two different polarization-dependent measurements(I_(zzz) and I_(zxx), or equivalently I_(ppp) and I_(pss). In this case,χ_(zzz) ⁽²⁾ can be determined by measuring the p-polarized SHG signalusing p-polarized fundamental excitation light. For example, iffundamental excitation light at 800 nm is used (e.g., from a Ti:Sapphire mode-locked laser), the second harmonic signal is detected at400 nm. In general, I_(ppp), which is the SHG signal intensity observedunder p-polarized excitation and p-polarized SHG detection, is governedby several components of the nonlinear susceptibily. However, asimplified approach for isolating only χ_(zzz) ⁽²⁾ in this measurementis achieved by measuring the SHG signal at the critical angle ofincidence in a total reflection geometry using a silica prism. In totalinternal reflection (TIR) geometry the measured SHG intensity isdetermined by the refractive indices of the prism and the buffer inwhich the surface-tethered proteins are bathed, since the off-axistensor components of χ⁽²⁾ vanish, leaving only χ_(zzz) ⁽²⁾ whichdetermines the measured I_(ppp) SHG signal intensity (referring topolarization of the fundamental/SHG beams). Similarly, χ_(zxx) ⁽²⁾ canbe determined by measuring I_(pss) using s-polarized fundamental lightand measuring the p-polarized SHG light intensity. In cases where N_(s)and α_(z′z′z′) are unknown, ratios of the intensities measured underdifferent polarization combinations can be used to eliminate theseparameters, leaving only ratios of the orientational distributionsthemselves which are trigonometric functions of θ, where θ is defined asthe mean angle between the z-axis in the molecular frame and the surfacenormal. When the orientational distribution is narrow, θ can bedetermined directly. By repeating the measurements of the SHG intensityunder different polarizations (e.g., I_(zzz) and I_(zxx)) using proteinlabeled at two or more different sites (e.g., in two or more differentsingle-site cysteine mutants, at these cysteine sites), one can obtain,for example, a different θ for each label site that can be used as aconstraint in structure determination. Each measurement requires alabeled protein, preferably with the label site-specifically attached(e.g., covalently attached via site-directed cysteine mutagenesis) at aknown position within the protein. By also varying the experimentalconditions to produce differently oriented protein relative to thesurface plane along with the two or more different sites of labeling,independent measurements can be made to determine multiple parametersdescribing the orientational distribution, thereby providing importantconstraints for protein structure determination. A key step of thepresent invention is to make measurements of a protein labeled at two ormore different sites (preferably in separate protein-label conjugates)under two or more different experimental conditions that result indifferent values of χ⁽²⁾ or ratios of χ⁽²⁾ components e.g., χ_(zzz)⁽²⁾/χ_(zxx) ⁽²⁾). By measuring values of χ⁽²⁾ for the same proteinlabeled at two or more different sites under two or more differentexperimental conditions, one can obtain more accurate measurements ofthe θ's (in the lab frame) and relate the difference between them, i.e.in the protein frame, to the structure of the protein.

For example, with two proteins each labeled at one site (site 1 and site2), each of which is oriented differently under different experimentalconditions on a surface, one can have four distinct measurements forwhich two polarization-dependent SHG intensities can be made as shownbelow, for a total of 8 independent intensity measurements:

Orientation 1: Protein labeled at site 1—measure I_(zzz), I_(zxx)

-   -   Protein labeled at site 2—measure I_(zzz), I_(zxx)

Orientation 2: Protein labeled at site 1—measure I_(zzz), I_(zxx)

-   -   Protein labeled at site 2—measure I_(zzz), I_(zxx)

By rationing the measured intensities of I_(zzz) and I_(zxx) and usingthe appropriate Fresnel factors, the hyperpolarizability and numberdensity of the SHG-active labels can be eliminated using methods wellknown to those of skill in the art. Because proteins are macromoleculeswith many potential label attachment sites, labeling different sites andorienting the protein in different ways offers a convenient way toobtain more than one independent ratio of polarization-dependentintensities (e.g., I_(zzz) and I_(zxx)), thereby allowing one to fit theorientational distribution term within the brackets of the Equations in(1) to multiple model parameters.

For example, if using three or more different sets of experimentalconditions which produce a different protein orientational distributionfor two separate protein conjugates labeled at two different cysteinesites, one obtains (2×3)−1=5 intensity ratios which can be used todetermine parameters of the molecular orientational distribution. Thedifferent 0's in the lab frame can be used to calculate theintramolecular angle differences between the two label sites in theprotein frame, i.e. to obtain structural information in the proteinframe. This is because although the protein will be oriented differentlyin the lab frame when it is tethered to a surface via a His-tag at theC-terminus vs. the N-terminus (resulting in different 0's andorientational distributions), the relationship between the labels in theprotein frame of reference should remain constant. Therefore, when theprotein is tethered in different orientational distributions to thesurface, the additional independent measurements allow one to determinethe inter-angle measurements in the protein frame reference moreaccurately. In effect, each set of experimental condition will reorientthe protein on the surface so that multiple independent measurements ofthe lab frame angle of the label hyperpolarizability projected on thez-axis can be obtained. Each independent measurement can be used toprovide an additional constraint in determining the structure in theprotein frame of reference. In effect, the mean angle differencesbetween the two cysteine sites should remain constant across theexperimental conditions, allowing for fitting of the orientationaldistribution to additional model parameters beyond the mean angle, suchas the width of the orientational distribution (e.g., the full width athalf maximum (FWHM) of a Gaussian distribution centered around the meanangle). Using this approach, structures of ligand-bound protein could bedetermined rapidly by making two or more independent measurements of thevalues of χ⁽²⁾ for different label sites and using these angularconstraints determined by SHG along with the unbound (apo) X-ray crystalstructure coordinates to determine the best ligand-bound structure atatomic resolution, without requiring X-ray crystal structuredetermination of the ligand-bound complex. Such ligands could be smallmolecules, small molecule fragments, peptides, proteins, antibodies,oligonucleotides, and in general any ligand Known to Those Skilled inthe Art.

Multiple Conformations—Equilibrium Orientational Distributions:

If a protein exists in an equilibrium of multiple conformational states,the protein will be described by a multi-modal (or multi-state)orientational distribution at each label site. If the distribution iscomposed of a sum of Gaussians with different weights, mean angles (θs)and distribution widths (Δs), a complete description of the protein'sconformational landscape will depend on determining each of theseparameters. For example, if the local structure of the protein at labelsite 1 adopts 3 conformations, under these assumptions, the localorientational distribution can be described by 3×3 parameters to bedetermined, or 9 unknowns, describing amplitude, mean angle, and widthfor each conformation. Label site 2 may adopt only 2 local conformationsand in that case can similarly be described by 6 parameters. For anexperimental geometry involving a single dominant tensor component ofthe hyperpolarizability, isotropic symmetry in the surface plane(azimuthal symmetry), and excitation at the critical angle, twoindependent components of χ⁽²⁾ can be determined for each experimentalcondition (e.g., χ_(zzz) ⁽²⁾ and χ_(zxx) ⁽²⁾ or χ_(zxx) ⁽²⁾); and N-1independent ratios of these values may be used to eliminate N_(s) andα_(z′z′z′)). Therefore, for an experiment involving two labeled mutantsunder the aforementioned assumptions, a minimum of 16 independentmeasurements (of the components of χ⁽²⁾) should be made (yielding 15independent ratios of χ⁽²⁾ values) which can be used to fit theorientational distributions. This approach assumes that the singledominant tensor component of the hyperpolarizability does not vary inmagnitude or phase with label site. The additional independentmeasurements, an aspect of the present invention, can be obtained byvarying the tag site (e.g., C- and N-terminus), tag length (e.g., 6×,8×, 10× and 12× His tags), buffer conditions (e.g., different saltconcentrations), and so on, or in general, any experimental conditionsthat varies the orientation of the protein on the surface and thusimpacts the measured values of χ⁽²⁾. All of these independentmeasurements can then be used, for example, in a global fitting methodto determine the solution-based conformational landscape (i.e.multi-state orientational distribution) in the protein frame ofreference at these two sites. In some embodiments, the X-ray crystalstructure coordinates of the protein may optionally be used as a furtherconstraint in the model building.

Absolute Polar Orientation Determination:

Although the tilt angle orientation of the label in the lab frame can bedetermined, this tilt angle is degenerate in two cones pointing towardand away from the surface, respectively. The present invention alsodiscloses a novel method for obtaining the absolute direction of thelabels, i.e. which direction the label points relative to the surfaceplane using a simple experiment. In this experiment, the SHG signalunder a given polarization condition is measured using: i) labeledprotein attached to an unlabeled surface; ii) unlabeled protein attachedto a labeled surface and iii) labeled protein attached to a labeledsurface. The labeled surface can be prepared in a variety of ways knownto those skilled in the art, for example through covalent carbodiimidecoupling of a carboxylated nonlinear-active label to anaminosilane-functionalized glass substrate surface. Alternatively, withsupported lipid bilayers (SLBs), one can covalently couple the samenonlinear-active label that attaches via amines or thiols (e.g.corresponding to lysine or cysteine residue side-chains) to a protein tothe bilayer doped with varying mole percentages of an amine orthiol-containing lipid. This surface then provides an SHG signal of itsown which, because the label is the same as that of the protein label,is in phase with the SHG signal generated from the protein. The labelattached to the supported bilayer has a known polar orientation byvirtue of its known directional coupling to the surface and its chemicalstructure. An experiment to determine the absolute polar orientation ofa label on a protein (and therefore potentially the polar orientation ofthe entire protein) can be carried out as follows. First, the SHG signalof the labeled surface in the absence of protein is measured (I_(L)).Second, the SHG signal of the labeled protein attached to the unlabeledsurface is measured (I_(P)). Third, the SHG signal of the surface ismeasured when labeled protein is attached to the labeled surface(I_(TOT)). The relationship between the different SHG signal is asfollows:I _(TOT) =I _(L) +I _(P)+sqrt(I _(L) ×I _(P))×cos(θ)

where cos(θ) describes the phase relationship (which flips in sign withthe absolute polar orientation toward or away from the surface) betweenthe labels attached to the protein molecules and the surface in thethird measurement (I_(TOT)). By measuring I_(TOT), I_(L), and I_(P)separately and comparing the measured signal intensities, the absolutepolar orientation of each label on the protein can be determined. Insome cases, e.g., when I_(L)+I_(P) are of roughly comparable magnitude,if I_(TOT) is smaller than I_(L) on its own, one can immediatelydetermine that destructive interference is occurring between the proteinlabel and the surface label; therefore, the labels are oriented inopposite polar orientations; if I_(TOT) is larger than I_(L) on its own,constructive interference is occurring and the labels are oriented inthe same polar direction. The magnitude of I_(L)+I_(P) can be varied bytuning, for example, the density of attachment sites on the supportedbilayer for the dye, or the density of proteins attached to the surface.

In some embodiments, the density of attachment sites on the supportedlipid bilayer may be varied by varying the percentage of a lipidcomponent of the bilayer that comprises an amine group or a thiol group(or any other functional group for which standard conjugationchemistries are available). In some embodiments, the percentage of thelipid component that comprises an amine or thiol group may range fromabout 0 percent to about 100 percent. In some embodiments, thepercentage of the lipid component that comprises an amine or thiol groupmay be at least 0 percent, at least 10 percent, at least 20 percent, atleast 30 percent, at least 40 percent, at least 50 percent, at least 60percent, at least 70 percent, at least 80 percent, at least 90 percent,or at least 100 percent. In some embodiments, the percentage of thelipid component that comprises an amine or thiol group may be at most100 percent, at most 90 percent, at most 80 percent, at most 70 percent,at most 60 percent, at most 50 percent, at most 40 percent, at most 30percent, at most 20 percent, or at most 10 percent. Those of skill inthe art will recognize that the percentage of the lipid component thatcomprises an amine group or a thiol group may have any value within thisrange, for example, about 12 percent.

In some embodiments, the density of nonlinear-active labeled proteinsattached to the surface may be varied by varying the concentration oflabeled protein in the solution that is incubated with the supportedlipid bilayer. For example, the concentration of a His tagged, labeledprotein may be varied in the solution that is incubated with a supportedlipid bilayer comprising a lipid component that further comprises aNi-NTA moiety. In some embodiment, the concentration of labeled proteinin the solution may range from about 1 nM to about 100 μM. In preferredembodiments, the concentration of labeled protein in the solution mayrange from about 100 nM to about 5 μM. In some embodiments, theconcentration of labeled protein in the solution may be at least 1 nM,at least 10 nM, at least 100 nM, at least 1 μM, at least 10 μM, or atleast 100 μM. In some embodiments, the concentration of labeled proteinin the solution may be at most 100 μM, at most 10 μM, at most 1 μM, atmost 100 nM, at most 10 nM, or at most 1 nM. Those of skill in the artwill recognize that the concentration of labeled protein in the solutionmay have any value with this range, for example, about 12 μM.

In some embodiments the density of nonlinear-active labeled protein onthe surface may be varied using any of a variety of techniques known tothose of skill in the art over the range of about 10² molecules/cm² toabout 10¹⁴ molecules/cm². In some embodiments, the density ofnonlinear-active labeled protein on the surface may be at least 10²molecules/cm², at least 10³ molecules/cm², at least 10⁴ molecules/cm²,at least 10⁵ molecules/cm², at least 10⁶ molecules/cm², at least 10⁷molecules/cm², at least 10⁸ molecules/cm², at least 10⁹ molecules/cm²,at least 10¹⁰ molecules/cm², at least 10¹¹ molecules/cm², at least 10¹²molecules/cm², at least 10¹³ molecules/cm², or at least 10¹⁴molecules/cm². In some embodiments, the density of nonlinear-activelabeled protein on the surface may be at most 10¹⁴ molecules/cm², atmost 10¹³ molecules/cm², at most 10¹² molecules/cm², at most 10¹¹molecules/cm², at most 10¹⁰ molecules/cm², at most 10⁹ molecules/cm², atmost 10⁸ molecules/cm², at most 10⁷ molecules/cm², at most 10⁶molecules/cm², at most 10⁵ molecules/cm², at most 10⁴ molecules/cm², atmost 10³ molecules/cm², or at most 10² molecules/cm². Those of skill inthe art will recognize that the density of nonlinear-active labeledprotein on the surface may have any value within this range, forexample, about 4.0×10¹² molecules/cm².

Background Signal Subtraction:

If background SHG signal is present due to the substrate-bufferinterface, for example, this can be “subtracted” out in various ways.For example, the phase difference between the SHG signal from the labelon the protein and the SHG signal due to the background can be measuredin an interferometric experiment such as the one described in Reider,G., et al. (1999), “Coherence Artifacts in Second Harmonic Microscopy”,Applied Physics B-Lasers and Optics 68, 343-347. The SHG signal due tothe protein alone can then be determined.

Electric Field Orientation, Strength and Characteristics:

An electric field can be applied to manipulate the orientation of thebiomolecules in the lab frame at the interface. The electric fielddirection can be across the surface, perpendicular to it, or in general,at any angle relative to the surface plane. In one embodiment, oneelectrode is placed underneath a lipid bilayer membrane or other surfacechemistry for protein attachment to the substrate, e.g. a glasssubstrate. A counter-electrode is placed above the substrate surfaceplane, for example at the top of the liquid in a sample well. In anotherembodiment, two or more electrodes are placed in the substrate surfaceplane and the electric field direction is parallel to thesubstrate-membrane interface.

In another embodiment, an array of electrodes can be placed around thetethered or immobilized protein sample, as illustrated in FIG. 9. Forexample, a circular array of electrodes can be placed parallel to themembrane interface on a glass substrate surface, each spaced about 10degrees apart from each other. Voltage applied to a pair of electrodesthat are 180 degrees apart from each other allows the azimuthaldirection of the electric field to be changed at will and in a rapidfashion. For example, the azimuthal direction of the electric field canbe swept around the entire circle in a second or a fraction of a second.

Electrodes may be patterned on the substrate surface using any of avariety of techniques known to those of skill in the art. Examplesinclude, but are not limited to, screen printing, photolithographicpatterning, sputter coating, chemical vapor deposition, or anycombination thereof.

Electrodes may be fabricated from any of a variety of material, as iswell known to those of skill in the art. Examples of suitable electrodematerials include, but are not limited to, silver, gold, platinum,copper, aluminum, graphite, indium tin oxide (ITO), semiconductormaterials, conductive polymers, or any combination thereof.

In some embodiments, it may be desirable to passivate the surface of oneor more electrodes, e.g., to minimize corrosion of the electrodesurfaces that are in contact with aqueous buffers, and/or to preventcontamination of or interference with proteins or other biologicalcomponents, and/or to prevent current flow in the sample. Any of avariety of passivation techniques known to those of skill in the art maybe used, and will in general depend on the choice of materials used tofabricate the electrode(s). For example, indium tin oxide electrodes onglass substrates may be passivated by growth or deposition of a 30 nmSiO₂ layer. Metal or semiconductor electrodes will often develop aninert “native oxide” layer upon exposure to air that may serve as apassivation layer. This inert surface layer is usually an oxide or anitride, with a thickness of a monolayer (1-3 Å) for platinum, about 15Å for silicon, and may be close to 50 Å thick for aluminum after longexposures to air.

The electric field can be DC or AC, i.e. time-invariant or time-varying.In the latter case, it can take a sinusoidal wave of any frequency or itcan be a complex wave (e.g., a step function, a saw tooth pattern, etc.)comprised of many frequency components, and the field can oscillatebetween positive or negative values or remain all positive or allnegative. Non-periodic or pulsed electric fields can also be applied insome embodiments. The SHG signal can be read before, during or afterapplication of an electric field to the sample.

In some embodiments, the electric field strength may range from aboutzero to about 10⁶ V/cm, or larger. In some embodiments, the electricfield strength may be at least zero, at least 10 V/cm, at least 10²V/cm, at least 10³ V/cm, at least 10⁴ V/cm, at least 10⁵ V/cm, or atleast 10⁶ V/cm. In some embodiments, the electric field strength may beat most 10⁶ V/cm, at most 10⁵ V/cm, at most 10⁴ V/cm, at most 10³ V/cm,at most 10² V/cm, at most 10 V/cm. Those of skill in the art willrecognize that the electric field strength may have any value withinthis range, for example, about 500 V/cm.

In some embodiments, the frequency at which the electric field is variedmay range from about 0 Hz to about 10⁵ Hz. In some embodiments, thefrequency at which the electric field is varied may be at least 0 Hz, atleast 10 Hz, at least 10² Hz, at least 10³ Hz, at least 10⁴ Hz, or atleast 10⁵ Hz. In some embodiments, the frequency at which the electricfield is varied may be at most 10⁵ Hz, at most 10⁴ Hz, at most 10³ Hz,at most 10² Hz, or at most 10 Hz. Those of skill in the art willrecognize that the frequency at which the electric field is varied mayhave any value within this range, for example, about 125 Hz.

The electric field can be used to manipulate the orientation of theprotein molecules and thus the baseline signal or SHG polarizationdependence. In some embodiments, if orientational isotropy in thesubstrate surface plane (i.e. the XY plane) occurs in the absence of anapplied electric field and is preserved when a field is applied, onlytwo or three independent non-vanishing components of the nonlinearsusceptibility (χ⁽²⁾) will exist. In other embodiments in whichorientational anisotropy is present in the surface plane, either before,during or after application of an electric field, more than two or threeindependent, non-vanishing components of χ⁽²⁾ will exist, allowing foradditional independent SHG measurements with different combinations ofpolarized fundamental and second-harmonic light. In some embodiments inwhich orientational anisotropy exists at the surface plane (e.g., at alipid biomembrane to which labeled proteins are attached), multipleindependent measurements of the χ⁽²⁾ can be made at different azimuthalangles. For example, if an electric field is applied parallel to thesurface, and this causes a change in the orientational distribution ofthe protein molecules from isotropic in-plane to anisotropic in-plane,additional independent optical measurements can be made in manyazimuthal directions relative to the direction of the applied electricfield to determine the molecular orientational distribution.

Optical Multiwell Plate with Integrated Electrodes:

As will be discussed in more detail below, in some embodiments the SHGmeasurements described herein are preferentially performed using amicrowell plate format. In some embodiments using a 384-well plate (orother microwell plate or multi-chamber formats), electrodes can bepatterned on a substrate surface inside of and adjacent to the walls ofthe wells, as part of a lid that is used to seal the wells, elsewhere onthe substrate surface (which may be glass) within the wells, or anywherethat allows both application of voltage to produce an electric field onthe sample and optical reading of the SHG signal.

Optical Multiwell Plate with Hemispherical Prisms:

In some embodiments, particularly in cases in which an anisotropicorientational distribution of the molecules exists at the surface, itwill be useful to optically probe the sample at different azimuthaldirections relative to the anisotropic axis. As an alternative torotating the sample relative to the optical axis, the optical axis canbe rotated relative to the fixed sample. To accomplish this,hemispherical prisms placed at the bottom of, or near each well, can beused to direct incoming light incident on the prisms at arbitrary anglesrelative to the well to the interfacial region containing the molecules.In some embodiments, the hemispherical prisms are bonded to orintegrated with the substrate in a glass-bottom multi-well plate, asillustrated in FIG. 8. The hemispherical prisms make optical contactwith the multi-well plate, thereby permitting transmission of theoptical beam with minimal loss. In some embodiments, the opticalmulti-well plate will comprise a 384-well glass-bottom plate or otherglass-bottom microwell plate format (i.e. standard microwell plateformats that are well known to those of skill in the art).

In some embodiments, the microwell plate device comprising an array ofhemispherical prisms bonded to or integrated with the glass substratethat forms the bottom of the wells may further comprise a patternedarray of electrodes on the upper surface of the glass substrate withineach well so that polarized SHG measurements may be made while applyingelectric fields of different field strengths.

Measurement of conformational change: In one embodiment, aligand-induced conformational change is measured at one or more labelsites within the protein. In one embodiment, single-site cysteineresidues are used. Combinations of polarized fundamental and nonlinearlight are used to determine the components of χ⁽²⁾ before and afterligand addition. For example, if the biomolecules are orientedisotropically on the surface and the molecular hyperpolarizability isdominated by a single tensor element (e.g., α_(z′z′z′), or equivalentlyin some literature β_(z′z′z′)) then 4 independent measurements of χ⁽²⁾can be made: two before and two after contacting the biomolecules withthe ligand: (e.g., χ_(zzz) and χ_(xzx) or χ_(zxx)). If one models theorientational distribution before and after conformational change ascontaining three adjustable parameters (θ₁, θ₂, and σ) where θ₁ is theaverage lab frame orientation prior to conformational change, θ₂ is theaverage lab frame orientation after conformational change, and a is theorientational width of a Gaussian around both θ₁ and θ₂, i.e. in onenon-limiting example of a model assumption the width of theorientational angle distribution remains the same after theconformational change, then one can determine these three parameters bydetermining the three independent ratios of the four components of χ⁽²⁾(χ¹ _(zzz), χ¹ _(zxx), χ² _(zzz), χ² _(zxx)): e.g., χ¹ _(zzz). χ¹_(zxx), χ² _(zzz)/χ_(zxx), χ¹ _(zzz)/χ² _(zzz) where the superscripts 1and 2 indicate the conformations before and after the ligand-inducedchange, respectively. The mean angles (θ₁ and θ₂) determined from suchmeasurements and equation 1 can be used to determine the angular changein label orientation at the label site in the protein frame of referencedue to conformational change. This process can be repeated for anynumber of label sites using different single-site cysteine mutants, anda model of local or the global ligand-bound structure can be determined.In some embodiments, the model optionally incorporates the X-ray crystalstructure coordinates or other structural constraints (e.g., from NMRdata, small angle X-ray scattering data, or any other measurements knownto those skilled in the art).

High Throughput Systems and Methods

Systems and methods are disclosed herein for implementing highthroughput analysis of structure of conformation in biological entitiesbased on the use of second harmonic generation or related nonlinearoptical detection techniques. As used herein, “high throughput” is arelative term used in comparison to structural measurements performedusing traditional techniques such as NMR or X-ray crystallography. Aswill be described in more detail below, the SHG-based methods andsystems disclosed herein are capable of performing structuraldeterminations at a rate that is at least an order-of-magnitude fasterthan these conventional techniques.

In one aspect, this disclosure provides a method for high throughputdetection of conformation or conformational change in one or morebiological entities, the method comprising (i) labeling one or moretarget biological entities, e.g. protein molecules, with anonlinear-active label or tag, (ii) tethering or immobilizing the one ormore labeled target biological entities at one or more discrete regionsof a planar substrate surface, wherein the substrate surface furthercomprises an optical interface, (iii) sequentially exposing eachdiscrete region to excitation light by changing the position of thesubstrate relative to an external light source, (iv) collecting anonlinear optical signal emitted from each discrete region as it isexposed to excitation light, and (v) processing said nonlinear opticalsignal to determine an orientation, conformation, or conformationalchange of each of the one or more biological entities. In anotheraspect, the method further comprises (vi) contacting each of the one ormore biological entities with one or more test entities following thefirst exposure to excitation light, (vii) subsequently re-exposing eachdiscrete region to excitation light one or more times, (viii) collectinga nonlinear optical signal from each discrete region as it is exposed toexcitation light, and (ix) processing said nonlinear optical signals todetermine whether or not a change in orientation or conformation hasoccurred in the one or more biological entities as a result ofcontacting with said one or more test entities. In one aspect of themethod, nonlinear optical signals are detected only once followingcontact of the one or more biological entities with one or more testentities (i.e. endpoint assay mode), and then used to determine whetheror not conformational change has occurred. In another aspect, nonlinearoptical signals are collected repeatedly and at defined time intervalsfollowing contact of the one of more biological entities with one ormore test entities (i.e. kinetics mode), and then used to determine thekinetics of conformational change in the one or more biologicalentities. In a preferred aspect of the method, each discrete region ofthe substrate comprises a supported lipid bilayer structure, andbiological entities are immobilized in each discrete region by means oftethering to or embedding in the lipid bilayer. In another preferredaspect of the method, the excitation light is delivered to the substratesurface, i.e. the optical interface, by means of total internalreflection, and the nonlinear optical signals emitted from the discreteregions of the substrate surface are collected along the same opticalaxis as the reflected excitation light.

In order to implement high throughput analysis of protein structure orconformational change using nonlinear optical detection, the systemsdescribed herein require several components (illustrated schematicallyin FIG. 4), including (i) at least one suitable excitation light sourceand optics for delivering the at least one excitation light beam to anoptical interface, (ii) an interchangeable substrate comprising theoptical interface, to which one or more biological entities have beentethered or immobilized in discrete regions of the substrate, (iii) ahigh-precision translation stage for positioning the substrate relativeto the at least one excitation light source, and (iv) optics forcollecting nonlinear optical signals generated as a result ofilluminating each of the discrete regions of the substrate withexcitation light and delivering said nonlinear signals to a detector,and (v) a processor for analyzing the nonlinear optical signal datareceived from the detector and determining conformation orconformational change for the one or more biological entitiesimmobilized on the substrate. In some aspects, the systems and methodsdisclosed herein further comprise the use of (vi) a programmablefluid-dispensing system for delivering test entities to each of thediscrete regions of the substrate, and (vii) the use of plate-handlingrobotics for automated positioning and replacement of substrates at theinterface with the optical system.

The methods and systems disclosed herein may be configured for analysisof a single biological entity contacted with a plurality of testentities, or for analysis of a plurality of biological entitiescontacted with a single test entity, or any combination thereof. Whencontacting one or more biological entities with a plurality of testentities, the contacting step may be performed sequentially, i.e. byexposing the immobilized biological entity to a single test entity for aspecified period of time, followed by an optional rinse step to removethe test entity solution and regenerate the immobilized biologicalentity prior to introducing to the next test entity, or the contactingstep may be performed in parallel, i.e. by having a plurality ofdiscrete regions comprising the same immobilized biological entity, andexposing the biological entity in each of the plurality of discreteregions to a different test entity. The methods and systems disclosedherein may be configured to perform analysis of conformational change inat least one biological entity, at least two biological entities, atleast four biological entities, at least six biological entities, atleast eight biological entities, at least ten biological entities, atleast fifteen biological entities, or at least twenty biologicalentities. In some aspects, methods and systems disclosed herein may beconfigured to perform analysis of conformational change in at mosttwenty biological entities, at most fifteen biological entities, at mostten biological entities, at most eight biological entities, at most sixbiological entities, at most four biological entities, at most twobiological entities, or at most one biological entity. Similarly, themethods and systems disclosed herein may be configured to performanalysis of conformational change upon exposure of the one or morebiological entities to at least 1 test entity, at least 5 test entities,at least 10 test entities, at least 50 test entities, at least 100 testentities, at least 500 test entities, at least 1,000 test entities, atleast 5,000 test entities, at least 10,000 test entities, or at least100,000 test entities. In some aspects, the methods and systemsdisclosed herein may be configured to perform analysis of conformationalchange upon exposure of the one or more biological test entities to atmost 100,000 test entities, at most 10,000 test entities, at most 5,000test entities, at most 1,000 test entities, at most 500 test entities,at most 100 test entities, at most 50 test entities, at most 10 testentities, at most 5 test entities, or at most 1 test entity.

Biological Entities and Test Entities

As used herein, the phrase “biological entities” comprises but is notlimited to cells, proteins, peptides, receptors, enzymes, antibodies,DNA, RNA, biological molecules, oligonucleotides, solvents, smallmolecules, synthetic molecules, carbohydrates, or any combinationthereof. Similarly, the phrase “test entities” also comprises but is notlimited to cells, proteins, peptides, receptors, enzymes, antibodies,DNA, RNA, biological molecules, oligonucleotides, solvents, smallmolecules, synthetic molecules, carbohydrates, or any combinationthereof. In some aspects, biological entities may comprise drug targets,or portions thereof, while test entities may comprise drug candidates,or portions thereof.

Nonlinear-Active Labels and Labeling Techniques

As noted above, most biological molecules are not intrinsicallynonlinear-active. Exceptions include collagen, a structural protein thatis found in most structural or load-bearing tissues. SHG microscopy hasbeen used extensively in studies of collagen-containing structures, forexample, the cornea. Other biological molecules or entities must berendered nonlinear-active by means of introducing a nonlinear-activemoiety such as a tag or label. A label for use in the present inventionrefers to a nonlinear-active moiety, tag, molecule, or particle whichcan be bound, either covalently or non-covalently to a molecule,particle or phase (e.g., a lipid bilayer) in order to render theresulting system more nonlinear optical active. Labels can be employedin the case where the molecule, particle or phase (e.g., lipid bilayer)is not nonlinear active to render the system nonlinear-active, or with asystem that is already nonlinear-active to add an extra characterizationparameter into the system. Exogenous labels can be pre-attached to themolecules, particles, or other biological entities, and any unbound orunreacted labels separated from the labeled entities before use in themethods described herein. In a specific aspect of the methods disclosedherein, the nonlinear-active moiety is attached to the target moleculeor biological entity in vitro prior to immobilizing the target moleculesor biological entities in discrete regions of the substrate surface. Thelabeling of biological molecules or other biological entities withnonlinear-active labels allows a direct optical means of detectinginteractions between the labeled biological molecule or entity andanother molecule or entity (i.e. the test entity) in cases where theinteraction results in a change in orientation or conformation of thebiological molecule or entity using a surface-selective nonlinearoptical technique.

In alternative aspects of the methods and systems described herein, atleast two distinguishable nonlinear-active labels are used. Theorientation of the attached two or more distinguishable labels wouldthen be chosen to facilitate well defined directions of the emanatingcoherent nonlinear light beam. The two or more distinguishable labelscan be used in assays where multiple fundamental light beams at one ormore frequencies, incident with one or more polarization directionsrelative to the optical interface are used, with the resulting emanationof at least two nonlinear light beams. In some embodiments, the numberof distinguishable nonlinear-active labels used my be at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, or at least ten.

Examples of nonlinear-active tags or labels include, but are not limitedto, the compounds listed in Table 1, and their derivatives.

TABLE 1 Examples of Nonlinear-Active Tags 2-aryl-5-(4-pyridyl)oxazole2-(4-pyridyl)-cycloalkano[d]oxazoles 5-aryl-2-(4-pyridyl)oxazole7-Hydroxycoumarin-3-carboxylic acid, succinimidyl ester Azo dyesBenzooxazoles Bithiophenes Cyanines Dapoxyl carboxylic acid,succinimidyl ester Diaminobenzene compounds Diazostilbenes FluoresceinsHemicyanines lndandione-1,3-pyidinium betaine lndodicarbocyaninesMelamines Merocyanines Methoxyphenyl)oxa-zol-2-yl)pyridinium bromide)Methylene blue Oxazole or oxadizole molecules Oxonols PerylenesPhenothiazine-stilbazole Polyenes Polyimides Polymethacrylates PyMPO(pyridyloxazole) PyMPO, succinimidyl ester(1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-PyMPO, maleimideStilbazims Stilbenes Stryryl-based dyes Sulphonyl-substitutedazobenzenes Thiophenes Tricyanovinyl aniline Tricyanovinyl azo

In evaluating whether a species may be nonlinear-active, the followingcharacteristics can indicate the potential for nonlinear activity: alarge difference dipole moment (difference in dipole moment between theground and excited states of the molecule), a large Stokes shift influorescence, or an aromatic or conjugated bonding character. In furtherevaluating such a species, an experimenter can use a simple techniqueknown to those skilled in the art to confirm the nonlinear activity, forexample, through detection of SHG from an air-water interface on whichthe nonlinear-active species has been distributed. Once a suitablenonlinear-active species bas been selected for the experiment at hand,the species can be conjugated, if desired, to a biological molecule orentity for use in the surface-selective nonlinear optical methods andsystems disclosed herein.

The following reference and references therein describe techniquesavailable for creating a labeled biological entity from a synthetic dyeand many other molecules: Greg T. Hermanson, Bioconjugate Techniques,Academic Press, New York, 1996.

In a specific aspect of the methods and systems disclosed, metalnanoparticles and assemblies thereof are modified to create biologicalnonlinear-active labels. The following references describe themodification of metal nanoparticles and assemblies: J. P. Novak and D.L. Feldheim, “Assembly of Phenylacetylene-Bridged Silver and GoldNanoparticle Arrays”, J. Am. Chem. Soc. 122:3979-3980 (2000); J. P.Novak, et al., “Nonlinear Optical Properties of Molecularly Bridged GoldNanoparticle Arrays”, J. Am. Chem. Soc. 122:12029-12030 (2000); Vance,F. W., Lemon, B. I., and Hupp, J. T., “Enormous Hyper-RayleighScattering from Nanocrystalline Gold Particle Suspensions”, J. Phys.Chem. B 102:10091-93 (1999).

In yet another aspect of the methods and systems disclosed herein, thenonlinear activity of the system can also be manipulated through theintroduction of nonlinear analogues to molecular beacons, that is,molecular beacon probes that have been modified to incorporate anonlinear-active label (or modulator thereof) instead of fluorophoresand quenchers. These nonlinear optical analogues of molecular beaconsare referred to herein as molecular beacon analogues (MB analogues orMBA). The MB analogues to be used in the described methods and systemscan be synthesized according to procedures known to one of ordinaryskill in the art.

Types of Biological Interactions Detected

In addition to determining orientation or structure of proteins andother biological molecules, the methods and systems disclosed hereinprovide for detection of a variety of interactions between biologicalentities, or between biological entities and test entities, depending onthe choice of biological entities, test entities, and non-linear activelabeling technique employed. In one aspect, the present disclosureprovides for the qualitative detection of binding events, e.g. thebinding of a ligand to a receptor, as indicated by the resultingconformational change induced in the receptor. In another aspect, thepresent disclosure provides for quantitative analysis of binding events,e.g. the binding of a ligand to a receptor, by performing replicatemeasurements using different concentrations of the ligand molecule andgenerating a dose-response curve using the percent change in maximalconformational change observed. Similarly, other aspects of the presentdisclosure may provide methods for qualitative or quantitativemeasurements of enzyme-inhibitor interactions, antibody-antigeninteractions, the formation of complexes of biological macromolecules,or interactions of receptors with allosteric modulators.

In other specific embodiments, MB analogues can be used according to themethods disclosed herein as hybridization probes that can detect thepresence of complementary nucleic acid target without having to separateprobe-target hybrids from excess probes as in solution-phasehybridization assays, and without the need to label the targetsoligonucleotides. MB analogue probes can also be used for the detectionof RNAs within living cells, for monitoring the synthesis of specificnucleic acids in sample aliquots drawn from bioreactors, and for theconstruction of self-reporting oligonucleotide arrays. They can be usedto perform homogeneous one-well assays for the identification ofsingle-nucleotide variations in DNA and for the detection of pathogensor cells immobilized to surfaces for interfacial detection.

Interactions between biological entities or biological and test entities(e.g. binding reactions, conformational changes, etc.) can be correlatedthrough the methods presently disclosed to the following measurablenonlinear signal parameters: (i) the intensity of the nonlinear light,(ii) the wavelength or spectrum of the nonlinear light, (iii) thepolarization of the nonlinear light, (iv) the time-course of (i), (ii),or (iii), and/or vi) one or more combinations of (i), (ii), (iii), and(iv).

Laser Light Sources and Excitation Optical System

FIG. 5 illustrates one aspect of the methods and systems disclosedherein wherein second harmonic light is generated by reflecting incidentfundamental excitation light from the surface of a substrate comprisingthe sample interface (or optical interface). In some embodiments, thesubstrate is optically-coupled to a prism used to deliver laser light atthe appropriate angle to induce total internal reflection at thesubstrate surface (FIG. 6). In some embodiments, the optical coupling isprovided by use of a thin film of an index-matching fluid. A laserprovides the fundamental light necessary to generate second harmoniclight at the sample interface. Typically this will be a picosecond orfemtosecond laser, either wavelength tunable or not tunable, andcommercially available (e.g. a Ti:Sapphire femtosecond laser or fiberlaser system). Light at the fundamental frequency (w) exits the laserand its polarization is selected using, for example a half-wave plateappropriate to the frequency and intensity of the light (e.g., availablefrom Melles Griot, Oriel, or Newport Corp.). The beam then passesthrough a harmonic separator designed to pass the fundamental light butblock nonlinear light (e.g. second harmonic light). This filter is usedto prevent back-reflection of the second harmonic beam into the lasercavity which can cause disturbances in the lasing properties. Acombination of mirrors and lenses are then used to steer and shape thebeam prior to reflection from a final mirror that directs the beam via aprism to impinge at a specific location and with a specific angle θ onthe substrate surface such that it undergoes total internal reflectionat the substrate surface. One of the mirrors in the optical path can bescanned if required using a galvanometer-controlled mirror scanner, arotating polygonal mirror scanner, a Bragg diffractor, acousto-opticdeflector, or other means known in the art to allow control of amirror's position. The substrate comprising the optical interface andnonlinear-active sample surface can be mounted on an x-y translationstage (computer controlled) to select a specific location on thesubstrate surface for generation of the second harmonic beam. In someaspects of the methods and systems presently described, it is desirableto scan or rotate one mirror in order to slightly vary the angle ofincidence for total internal reflection, and thereby maximize thenonlinear optical signal emitted from the discrete regions of thesubstrate surface without substantially changing the position of theilluminating excitation light spot. In some aspects, two (or more)lasers having different fundamental frequencies may be used to generatesum frequency or difference frequency light at the optical interface onwhich the non-linear active sample is immobilized.

Substrate Formats, Optical Interface, and Total Internal Reflection

As described above, the systems and methods of the present disclosureutilize a planar substrate for tethering or immobilization of one ormore biological entities on a top surface of the substrate, wherein thetop substrate surface further comprises the optical interface (or sampleinterface) used for exciting nonlinear optical signals. The substratecan be glass, silica, fused-silica, plastic, or any other solid materialthat is transparent to the fundamental and second harmonic light beams,and that supports total internal reflection at the substrate/sampleinterface when the excitation light is incident at an appropriate angle.In some aspects of the invention, the discrete regions within whichbiological entities are contained are configured as one-dimensional ortwo-dimensional arrays, and are separated from one another by means of ahydrophobic coating or thin metal layer. In other aspects, the discreteregions may comprise indents in the substrate surface. In still otheraspects, the discrete regions may be separated from each other by meansof a well-forming component such that the substrate forms the bottom ofa microwell plate (or microplate), and each individual discrete regionforms the bottom of one well in the microwell plate. In one aspect ofthe present disclosure, the well-forming component separates the topsurface of the substrate into 96 separate wells. In another aspect, thewell-forming component separates the top surface of the substrate into384 wells. In yet another aspect, the well-forming component separatesthe top surface of the substrate into 1,536 wells. In all of theseaspects, the substrate, whether configured in a planar array, indentedarray, or microwell plate format, may comprise a disposable orconsumable device or cartridge that interfaces with other optical andmechanical components of the high throughput system.

The methods and systems disclosed herein further comprise specifying thenumber of discrete regions or wells into which the substrate surface isdivided, irrespective of how separation is maintained between discreteregions or wells. Having larger numbers of discrete regions or wells ona substrate may be advantageous in terms of increasing the sampleanalysis throughput of the method or system. In one aspect of thepresent disclosure, the number of discrete regions or wells persubstrate is between 10 and 1,600. In other aspects, the number ofdiscrete regions or wells is at least 10, at least 20, at least 50, atleast 100, at least 200, at least 300, at least 400, at least 500, atleast 750, at least 1,000, at least 1,250, at least 1,500, or at least1,600. In yet other aspects of the disclosed methods and systems, thenumber of discrete regions or wells is at most 1,600, at most 1,500, atmost 1,000, at most 750, at most 500, at most 400, at most 300, at most200, at most 100, at most 50, at most 20, or at most 10. In a preferredaspect, the number of discrete regions or wells is 96. In anotherpreferred aspect, the number of discrete regions or wells is 384. In yetanother preferred aspect, the number of discrete regions or wells is1,536. Those of skill in the art will appreciate that the number ofdiscrete regions or wells may fall within any range bounded by any ofthese values (e.g. from about 12 to about 1,400).

The methods and systems disclosed herein also comprise specifying thesurface area of the discrete regions or wells into which the substratesurface is divided, irrespective of how separation is maintained betweendiscrete regions or wells. Having discrete regions or wells of largerarea may facilitate ease-of-access and manipulation of the associatedbiological entities in some cases, whereas having discrete regions orwells of smaller area may be advantageous in terms of reducing assayreagent volume requirements and increasing the sample analysisthroughput of the method or system. In one aspect of the presentdisclosure, the surface area of the discrete regions or wells is between1 mm² and 100 mm². In other aspects, the area of the discrete regions orwells is at least 1 mm², at least 2.5 mm², at least 5 mm², at least 10mm², at least 20 mm², at least 30 mm², at least 40 mm², at least 50 mm²,at least 75 mm², or at least 100 mm². In yet other aspects of thedisclosed methods and systems, the area of the discrete regions or wellsis at most 100 mm², at most 75 mm², at most 50 mm², at most 40 mm², atmost 30 mm², at most 20 mm², at most 10 mm², at most 5 mm², at most 2.5mm², or at most 1 mm². In a preferred aspect, the area of discreteregions or wells is about 35 mm². In another preferred aspect, the areaof the discrete regions or wells is about 8.6 mm². Those of skill in theart will appreciate that the area of the discrete regions or wells mayfall within any range bounded by any of these values (e.g. from about 2mm² to about 95 mm²).

Discrete regions of the substrate surface are sequentially exposed to(illuminated with) excitation light by re-positioning the substraterelative to the excitation light source. Total internal reflection ofthe incident excitation light creates an “evanescent wave” at the sampleinterface, which excites the nonlinear-active label and results ingeneration of second harmonic light (or in some aspects, sum frequencyor difference frequency light). Because the intensity of the evanescentwave, and hence the intensity of the nonlinear optical signalsgenerated, is dependent on the incident angle of the excitation lightbeam, precise orientation of the substrate plane with respect to theoptical axis of the excitation beam and efficient optical coupling ofthe beam to the substrate is critical for achieving optimal SHG signalacross the array of discrete regions. In some aspects of the presentdisclosure, total internal reflection is achieved by means of a singlereflection of the excitation light from the substrate surface. In otheraspects, the substrate may be configured as a waveguide such that theexcitation light undergoes multiple total internal reflections as itpropagates along the waveguide. In yet other aspects, the substrate maybe configured as a zero-mode waveguide, wherein an evanescent field iscreated by means of nanofabricated structures.

Efficient optical coupling between the excitation light beam and thesubstrate in an optical setup such as the one illustrated in FIGS. 5 and6 would typically be achieved by use of an index-matching fluid such asmineral oil, mixtures of mineral oil and hydrogenated terphenyls,perfluorocarbon fluids, glycerin, glycerol, or similar fluids having arefractive index near 1.5, wherein the index-matching fluid is wickedbetween the prism and the lower surface of the substrate. Since astatic, bubble-free film of index-matching fluid is likely to bedisrupted during fast re-positioning of the substrate, the systems andmethods disclosed herein include alternative approaches for creatingefficient optical coupling of the excitation beam to the substrate inhigh throughput systems.

FIG. 7 illustrates another aspect of a high throughput system of thepresent disclosure, in which a thin layer of index-matching elastomericmaterial is used in place of index-matching fluid to maintain efficientoptical coupling between the prism and substrate. In this case, thesubstrate is again packaged in a microwell plate format (e.g. a glassbottom microplate format), but with a thin layer of an index-matchingelastomeric material attached to or adjacent to the lower surface of thesubstrate, such that when placed in contact with the upper surface ofthe prism, the elastomer fills the gap between prism and substrate andprovides for efficient optical coupling. Examples of elastomericmaterials that may be used include, but are not limited to siliconeshaving a refractive index of about 1.4. In one aspect of the presentdisclosure, the refractive index of the elastomeric material is betweenabout 1.35 and about 1.6. In other aspects, the index of refraction isabout 1.6 or less, about 1.55 or less, about 1.5 or less, about 1.45 orless, about 1.4 or less, or about 1.35 or less. In yet other aspects,the index of refraction is at least about 1.35, at least about 1.4, atleast about 1.45, at least about 1.5, at least about 1.55, or at leastabout 1.6. Those of skill in the art will appreciate that the index ofrefraction of the elastomeric layer may fall within any range bounded byany of these values (e.g. from about 1.4 to about 1.6). In one aspect ofthis approach, the thickness of the layer of elastomeric material isbetween about 0.1 mm and 2 mm. In other aspects, the thickness of theelastomeric layer is at least 0.1 mm, at least 0.2 mm, at least 0.4 mm,at least 0.6 mm, at least 0.8 mm, at least 1.0 mm, at least 1.2 mm, atleast 1.4 mm, at least 1.6 mm, at least 1.8 mm, or at least 2.0 mm. Inanother aspect of this approach, the thickness of the elastomeric layeris at most 2.0 mm, at most 1.8 mm, at most 1.6 mm, at most 1.4 mm, atmost 1.2 mm, at most 1.0 mm, at most 0.8 mm, at most 0.6 mm, at most 0.4mm, at most 0.2 mm, or at most 0.1 mm. Those of skill in the art willappreciate that the thickness of the elastomeric layer my fall withinany range bounded by any of these values (e.g. from about 0.1 mm toabout 1.5 mm). In another aspect of this approach, the upper surface ofthe prism has a partially-cylindrical ridge or is domed (FIG. 7) tofocus the compression force and provide better contact betweensubstrate, elastomeric layer, and prism surface. This approach may alsorequire the use of a third axis of translation for positioning of thesubstrate, i.e. between excitation and detection steps, the substrate(microwell plate) would be raised slightly to eliminate contact betweenthe elastomeric layer and the prism prior to re-positioning thesubstrate to the location of the next discrete region to be analyzed.

FIGS. 10A-B and FIGS. 11A-B illustrate a preferred aspect of a highthroughput system of the present disclosure, in which an array of prismsor gratings is integrated with the lower surface of the substrate(packaged in a microwell plate format) and used to replace the fixedprism, thereby eliminating the need for index-matching fluids orelastomeric layers entirely. The array of prisms (or gratings) isaligned with the array of discrete regions or wells on the upper surfaceof the substrate in such a way that incident excitation light isdirected by an “entrance prism” (“entrance grating”) to a discreteregion or well that is adjacent to but not directly above the entranceprism (entrance grating), at an angle of incidence that enables totalinternal reflection of the excitation light beam from the sampleinterface (see FIG. 12), and such that the reflected excitation beam,and nonlinear-optical signals generated at the illuminated discreteregion, are collected by an “exit prism” (“exit grating”) that is againoffset from (adjacent to but not directly underneath) the discreteregion under interrogation, and wherein the entrance prism and exitprism (entrance grating and exit grating) for each discrete region aredifferent, non-unique elements of the array.

In general, for an array of discrete regions comprising M rows×N columnsof individual features, the corresponding prism or grating array willhave M+2 rows×N columns or N+2 columns×M rows of individual prisms orgratings. In some embodiments, M may have a value of at least 2, atleast 4, at least 6, at least 8, at least 12, at least 14, at least 16,at least 18, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, or at least 50 rows. In some embodiments, M mayhave a value of at most 50, at most 45, at most 40, at most 35, at most30, at most 25, at most 20, at most 18, at most 16, at most 14, at most12, at most 10, at most 8, at most 6, at most 4, or at most 2 rows.Similarly, in some embodiments, N may have a value of at least 2, atleast 4, at least 6, at least 8, at least 12, at least 14, at least 16,at least 18, at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, or at least 50 columns. In some embodiments, Nmay have a value of at most 50, at most 45, at most 40, at most 35, atmost 30, at most 25, at most 20, at most 18, at most 16, at most 14, atmost 12, at most 10, at most 8, at most 6, at most 4, or at most 2columns. As will be apparent to those of skill in the art, M and N mayhave the same value or different values, and may have any value withinthe range specified above, for example, M=15 and N=45.

The geometry and dimensions of the individual prisms or gratings,including the thickness of the prism or grating array layer, areoptimized to ensure that incident light undergoes total internalreflection at the selected discrete region of the substrate, andnonlinear optical signals generated at the selected discrete region arecollected, with high optical coupling efficiency, independently of theposition of substrate (microwell plate) relative to the excitation lightbeam. The prism or grating arrays may be fabricated by a variety oftechniques known to those of skill in the art, for example, in apreferred aspect, they may be injection molded from smooth flowing, lowbirefringence materials such as cyclic olefin copolymer (COC) or cyclicolefin polymer (COP), acrylic, polyester, or similar polymers. In someaspects, the prism or grating array may be fabricated as a separatecomponent, and subsequently integrated with the lower surface of thesubstrate. In other aspects, the prism or grating array may befabricated as an integral feature of substrate itself.

Immobilization Chemistries

As disclosed herein, substrates in any of the formats described aboveare further configured for immobilization of biological entities withinthe specified discrete regions. Immobilization of biological moleculesor cells may be accomplished by a variety of techniques known to thoseof skill in the art, for example, through the use of aminopropyl silanechemistries to functionalize glass or fused-silica surfaces with aminefunctional groups, followed by covalent coupling using amine-reactiveconjugation chemistries, either directly with the biological molecule ofinterest, or via an intermediate spacer or linker molecule. Non-specificadsorption may also be used directly or indirectly, e.g. through the useof BSA-NHS (BSA-N-hydroxysuccinimide) by first attaching a molecularlayer of BSA to the surface and then activating it withN,N′-disuccinimidyl carbonate. The activated lysine, aspartate orglutamate residues on the BSA react with surface amines on proteins.

In a preferred aspect of the present disclosure, biological moleculesmay be immobilized on the surface by means of tethering to or embeddingin “supported lipid bilayers”, the latter comprising small patches oflipid bilayer confined to a silicon or glass surface by means ofhydrophobic and electrostatic interactions, where the bilayer is“floating” above the substrate surface on a thin layer of aqueousbuffer. Supported phospholipid bilayers can also be prepared with orwithout membrane proteins or other membrane-associated components asdescribed, for example, in Salafsky et al., “Architecture and Functionof Membrane Proteins in Planar Supported Bilayers: A Study withPhotosynthetic Reaction Centers”, Biochemistry 35 (47): 14773-14781(1996); Gennis, R., Biomembranes, Springer-Verlag, 1989; Kalb et al.,“Formation of Supported Planar Bilayers by Fusion of Vesicles toSupported Phospholipid Monolayers”, Biochimica Biophysica Acta.1103:307-316 (1992); and Brian et al. “Allogeneic Stimulation ofCytotoxic T-cells by Supported Planar Membranes”, PNAS-BiologicalSciences 81(19): 6159-6163 (1984), relevant portions of which areincorporated herein by reference. Supported phospholipid bilayers arewell known in the art and there are numerous techniques available fortheir fabrication. Potential advantages of using supported lipidbilayers for immobilization of proteins or other biological entities onsubstrate surfaces or optical interfaces include (i) preservation ofmembrane protein structure for those proteins that typically span thecell membrane or other membrane components of cells and requireinteraction with the hydrophobic core of the bilayer for stabilizationof secondary and tertiary structure, (ii) preservation of twodimensional lateral and rotational diffusional mobility for studyinginteractions between protein components within the bilayer, and (iii)preservation of molecular orientation, depending on such factors as thetype of protein under study (i.e. membrane or soluble protein), how thebilayer membrane is formed on the substrate surface, and how the proteinis tethered to the bilayer (in the case of soluble proteins). Supportedbilayers, with or without tethered or embedded protein, should typicallybe submerged in aqueous solution to prevent their destruction whenexposed to air.

Soluble proteins and other biological entities may be tethered orattached to the supported lipid bilayer in an oriented fashion using anumber of different anchor molecules, linkers, and/or attachmentchemistries. As used herein, “anchor molecules” are molecules which areembedded in the lipid bilayer, and may comprise fatty acid,glycerolipid, glycerophospholipid, sphingolipid, or other lipid ornon-lipid molecules to which attachment moieties are conjugated.

Linker molecules are molecules used to provide spatial (“vertical”)separation between the attachment point of the protein or otherbiological entity being tethered and the attachment point on the anchormolecule embedded in the plane of the lipid bilayer. Examples ofsuitable linker molecules include, but are not limited to, omega-aminofatty acids, polyethylene glycols, and the like.

Attachment moieties (also referred to as “affinity tags”) are specificchemical structures or binding partners that provide for covalent ornon-covalent binding between two biological entities. Examples ofattachment moieties or affinity tags that are suitable for use in themethods disclosed herein include biotin and avidin (or biotin andstreptavidin), and His-tag/Ni-NTA binding partners.

The high affinity, non-covalent biotin-streptavidin interaction iswidely used in biological assay techniques to conjugate or immobilizeproteins or other biological entities. Biotinylation of proteins enablescapture by multivalent avidin or streptavidin molecules that arethemselves adhered to a surface (e.g. glass slides or beads) orconjugated to another molecule (e.g. through the use of abiotin-streptavidin-biotin bridge or linker). The biotin moiety issufficiently small that biotinylation typically doesn't interfere withprotein function. The high affinity (Kd of 10-14 M to 10-15M) and highspecificity of the binding interaction between biotin and avidin orstreptavidin enables capture of biotinylated proteins of interest evenfrom complex samples. Due to the extremely strong binding interaction,harsh conditions are needed to elute biotinylated protein fromstreptavidin-coated surfaces (typically 6M guanidine HCl at pH 1.5),which will often denature the protein of interest. The use of monomericforms of avidin or streptavidin, which have a decreased biotin-bindingaffinity of ˜10-8 M, may allow biotinylated proteins to be eluted withexcess free biotin if necessary. In the methods disclosed herein, lipidmolecules comprising biotin moieties may be incorporated into supportedlipid bilayers for the purpose of immobilizing or tethering biotinylatedproteins and/or other biotinylated biological entities to the bilayervia a biotin-avidin-biotin (or biotin-streptavidin-biotin) bridge.

Biotinylation of proteins and other biological entities may be performedby direct coupling, e.g. through conjugation of primary amines on thesurface of a protein using N-hydroxysuccinimidobiotin (NHS-biotin).Alternatively, recombinant proteins are conveniently biotinylated usingthe AviTag approach, wherein the AviTag peptide sequence(GLNDIFEAQKIEWHE) is incorporated into the protein through the use ofgenetic engineering and protein expression techniques. The presence ofthe AviTag sequence allows biotinylation of the protein by treatmentwith the BirA enzyme.

His tag chemistry is another widely used tool for purification ofrecombinant proteins and other biomolecules. In this approach, forexample, a DNA sequence specifying a string of six to nine histidineresidues may be incorporated into vectors used for production ofrecombinant proteins comprising 6×His or poly-His tags fused to their N-or C-termini. His-tagged proteins can then be purified and detected as aresult of the fact that the string of histidine residues binds toseveral types of immobilized metal ions, including nickel, cobalt andcopper, under specific buffer conditions. Supports such as agarose beadsor magnetic particles can be derivatized with chelating groups toimmobilize the desired metal ions, which then function as ligands forbinding and purification of the His-tagged biomolecules of interest.

The chelators most commonly used to create His-tag ligands arenitrilotriacetic acid (NTA) and iminodiacetic acid (IDA). Once NTA- orIDA-conjugated supports are prepared, they can be “loaded” with thedesired divalent metal (e.g., Ni, Co, Cu, or Fe). When using nickel asthe metal, for example, the resulting affinity support is usually calleda Ni-chelate, Ni-IDA or Ni-NTA support. Affinity purification ofHis-tagged fusion proteins is the most common application formetal-chelate supports in protein biology research. Nickel or cobaltmetals immobilized by NTA-chelation chemistry are the systems of choicefor this application. In the methods disclosed herein, lipid moleculescomprising Ni-NTA groups (or other chelated metal ions) may beincorporated into supported lipid bilayers for the purpose ofimmobilizing or tethering His-tagged proteins and other His-taggedbiological entities to the bilayer. In some embodiment, the supportedlipid bilayer may comprise 1,2-dioleoyl-sn-glycero-3-phosphocholine, andmay also contain1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiaceticacid)succinyl] (nickel salt) at various concentrations.

Poly-His tags bind best to chelated metal ions in near-neutral bufferconditions (physiologic pH and ionic strength). A typical binding/washbuffer consists of Tris-buffer saline (TBS) pH 7.2, containing 10-25 mMimidazole. The low-concentration of imidazole helps to preventnonspecific binding of endogenous proteins that have histidine clusters.Elution and recovery of captured His-tagged protein from chelated metalion supports, when desired, is typically accomplished using a highconcentration of imidazole (at least 200 mM), low pH (e.g., 0.1Mglycine-HCl, pH 2.5), or an excess of strong chelator (e.g., EDTA).Immunoglobulins are known to have multiple histidines in their Fc regionand can bind to chelated metal ion supports, therefore stringent bindingconditions (e.g. using an appropriate concentration of imidazole) arenecessary to avoid high levels of background binding if immunoglobulinsare present in a sample at high relative abundance compared to theHis-tagged proteins of interest. Albumins, such as bovine serum albumin(BSA), also have multiple histidines and can yield high levels ofbackground binding to chelated metal ion supports in the absence of moreabundant His-tagged proteins or the use of imidazole in the binding/washbuffer.

Collection Optics and Detector

FIG. 5 further illustrates the collection optics and detector used todetect nonlinear optical signals generated upon sequential illuminationof the discrete regions of the substrate. Because surface-selectivenonlinear optical techniques are coherent techniques, meaning that thefundamental and nonlinear optical light beams have wave fronts thatpropagate through space with well-defined spatial and phaserelationships, minimal collection optics are required. Emitted nonlinearoptical signals are collected by means of a prism (or the integratedprism or grating array of the microplate device described above) anddirected via a dichroic reflector and mirror to the detector. Additionaloptical components, e.g. lenses, optical bandpass filters, mirrors, etc.are optionally used to further shape, steer, and/or filter the beamprior to reaching the detector. A variety of different photodetectorsmay be used, including but not limited to photodiodes, avalanchephotodiodes, photomultipliers, CMOS sensors, or CCD devices.

X-Y Translation Stage

As illustrated in FIG. 4, implementation of the high throughput systemsdisclosed herein ideally utilizes a high precision X-Y (or in somecases, an X-Y-Z) translation stage for re-positioning the substrate (inany of the formats described above) in relation to the excitation lightbeam. Suitable translation stages are commercially available from anumber of vendors, for example, Parker Hannifin. Precision translationstage systems typically comprise a combination of several componentsincluding, but not limited to, linear actuators, optical encoders, servoand/or stepper motors, and motor controllers or drive units. Highprecision and repeatability of stage movement is required for thesystems and methods disclosed herein in order to ensure accuratemeasurements of nonlinear optical signals when interspersing repeatedsteps of optical detection and/or liquid-dispensing. Also, as the sizeof the focal spot for the excitation light [20-200 microns in diameteror on a side is substantially smaller than the size of the discreteregions on the substrate, in some aspects of the present disclosure, itmay also be desirable to return to a slightly different position withina given discrete region when making replicate measurements, or to slowlyscan the excitation beam across a portion of the discrete region overthe course of a single measurement, thereby eliminating potentialconcerns regarding the photo-bleaching effects of long exposures orprior exposures.

Consequently, the methods and systems disclosed herein further comprisespecifying the precision with which the translation stage is capable ofpositioning a substrate in relation to the excitation light beam. In oneaspect of the present disclosure, the precision of the translation stageis between about 1 um and about 10 um. In other aspects, the precisionof the translation stage is about 10 um or less, about 9 um or less,about 8 um or less, about 7 um or less, about 6 um or less, about 5 umor less, about 4 um or less, about 3 um or less, about 2 um or less, orabout 1 um or less. Those of skill in the art will appreciate that theprecision of the translation stage may fall within any range bounded byany of these values (e.g. from about 1.5 um to about 7.5 um).

Fluid Dispensing System

As illustrated in FIG. 4, some embodiments of the high throughputsystems disclosed herein further comprise an automated, programmablefluid-dispensing (or liquid-dispensing) system for use in contacting thebiological or target entities immobilized on the substrate surface withtest entities (or test compounds), the latter typically being dispensedin solutions comprising aqueous buffers with or without the addition ofa small organic solvent component, e.g. dimethylsulfoxide (DMSO).Suitable automated, programmable fluid-dispensing systems arecommercially available from a number of vendors, e.g. Beckman Coulter,Perkin Elmer, Tecan, Velocity 11, and many others. In a preferred aspectof the systems and methods disclosed herein, the fluid-dispensing systemfurther comprises a multichannel dispense head, e.g. a 4 channel, 8channel, 16 channel, 96 channel, or 384 channel dispense head, forsimultaneous delivery of programmable volumes of liquid (e.g. rangingfrom about 1 microliter to several milliliters) to multiple wells orlocations on the substrate.

Plate-Handling Robotics

In other aspects of the high throughput systems disclosed herein, thesystem further comprises a microplate-handling (or plate-handling)robotic system (FIG. 4) for automated replacement and positioning ofsubstrates (in any of the formats described above) in relation to theoptical excitation and detection optics, or for optionally movingsubstrates between the optical instrument and the fluid-dispensingsystem. Suitable automated, programmable microplate-handling roboticsystems are commercially available from a number of vendors, includingBeckman Coulter, Perkin Elemer, Tecan, Velocity 11, and many others. Ina preferred aspect of the systems and methods disclosed herein, theautomated microplate-handling robotic system is configured to movecollections of microwell plates comprising immobilized biologicalentities and/or aliquots of test compounds to and from refrigeratedstorage units.

Processor/Controller and Constraint-Based Scheduling Algorithm

In another aspect of the present disclosure, the high throughput systemsdisclosed further comprise a processor (or controller, or computersystem) (FIG. 4) configured to run system software which controls thevarious subsystems described (excitation and detection optical systems,X-Y (or X-Y-Z) translation stage, fluid-dispensing system, andplate-handling robotics) and synchronizes the different operationalsteps involved in performing high throughput conformational analysis. Inaddition to handling the data acquisition process, i.e. collection ofoutput electronic signals from the detector that correspond to thenonlinear optical signals associated with conformational change, theprocessor or controller is also typically configured to store the data,perform data processing and display functions (including determinationof whether or not changes in orientation or conformation have occurredfor the biological entities, or combinations of biological and testentities, that have been tested), and operate a graphical user interfacefor interactive control by an operator. The processor or controller mayalso be networked with other processors, or connected to the internetfor communication with other instruments and computers at remotelocations.

Typical input parameters for the processor/controller may include set-upparameters such as the total number of microwell plates to be analyzed;the number of wells per plate; the number of times excitation anddetection steps are to be performed for each discrete region of thesubstrate or well of the microplate (e.g. to specify endpoint assay orkinetic assay modes); the total timecourse over which kinetic datashould be collected for each discrete region or well; the order, timing,and volume of test compound solutions to be delivered to each discreteregion or well; the dwell time for collection and integration ofnonlinear optical signals; the name(s) of output data files; and any ofa number of system set-up and control parameters known to those skilledin the art.

In a preferred aspect of the present disclosure, the processor orcontroller is further configured to perform system throughputoptimization by means of executing a constraint-based schedulingalgorithm. This algorithm utilizes system set-up parameters as describedabove to determine an optimal sequence of interspersedexcitation/detection and liquid-dispensing steps for discrete regions orwells that may or may not be adjacent to each other, such that theoverall throughput of the system, in terms of number of biologicalentities and/or test entities analyzed per hour, is maximized.Optimization of system operational steps is an important aspect ofachieving high throughput analysis. In some aspects of the disclosedmethods and systems, the average throughput of the analysis system mayrange from about 10 test entities tested per hour to about 1,000 testentities tested per hour. In some aspects, the average throughput of theanalysis system may be at least 10 test entities tested per hour, atleast 25 test entities tested per hour, at least 50 test entities testedper hour, at least 75 test entities tested per hour, at least 100 testentities tested per hour, at least 200 test entities tested per hour, atleast 400 test entities tested per hour, at least 600 test entitiestested per hour, at least 800 test entities tested per hour, or at least1,000 test entities tested per hour. In other aspects, the averagethroughput of the analysis system may be at most 1,000 test entitiestested per hour, at most 800 test entities tested per hour, at most 600test entities tested per hour, at most 400 test entities tested perhour, at most 200 test entities tested per hour, at most 100 testentities tested per hour, at most 75 test entities tested per hour, atmost 50 test entities tested per hour, at most 25 test entities testedper hour, or at most 10 test entities tested per hour.

Computer Systems and Networks

In various embodiments, the methods and systems of the invention mayfurther comprise software programs installed on computer systems and usethereof. Accordingly, as noted above, computerized control of thevarious subsystems and synchronization of the different operationalsteps involved in performing high throughput conformational analysis,including data analysis and display, are within the bounds of theinvention.

The computer system 500 illustrated in FIG. 15 may be understood as alogical apparatus that can read instructions from media 511 and/or anetwork port 505, which can optionally be connected to server 509 havingfixed media 512. The system, such as shown in FIG. 15 can include a CPU501, disk drives 503, optional input devices such as keyboard 515 and/ormouse 516 and optional monitor 507. Data communication can be achievedthrough the indicated communication medium to a server at a local or aremote location. The communication medium can include any means oftransmitting and/or receiving data. For example, the communicationmedium can be a network connection, a wireless connection or an internetconnection. Such a connection can provide for communication over theWorld Wide Web. It is envisioned that data relating to the presentdisclosure can be transmitted over such networks or connections forreception and/or review by a party 522 as illustrated in FIG. 15.

FIG. 16 is a block diagram illustrating a first example architecture ofa computer system 100 that can be used in connection with exampleembodiments of the present invention. As depicted in FIG. 16, theexample computer system can include a processor 102 for processinginstructions. Non-limiting examples of processors include: the IntelXeon™ processor, the AMD Opteron™ processor, the Samsung 32-bit RISC ARM1176JZ(F)-S v1.0™ processor, the ARM Cortex-A8 Samsung S5PC100™processor, the ARM Cortex-A8 Apple A4™ processor, the Marvell PXA 930™processor, or a functionally-equivalent processor. Multiple threads ofexecution can be used for parallel processing. In some embodiments,multiple processors or processors with multiple cores can also be used,whether in a single computer system, in a cluster, or distributed acrosssystems over a network comprising a plurality of computers, cell phones,and/or personal data assistant devices.

As illustrated in FIG. 16, a high speed cache 104 can be connected to,or incorporated in, the processor 102 to provide a high speed memory forinstructions or data that have been recently, or are frequently, used byprocessor 102. The processor 102 is connected to a north bridge 106 by aprocessor bus 108. The north bridge 106 is connected to random accessmemory (RAM) 110 by a memory bus 112 and manages access to the RAM 110by the processor 102. The north bridge 106 is also connected to a southbridge 114 by a chipset bus 116. The south bridge 114 is, in turn,connected to a peripheral bus 118. The peripheral bus can be, forexample, PCI, PCI-X, PCI Express, or other peripheral bus. The northbridge and south bridge are often referred to as a processor chipset andmanage data transfer between the processor, RAM, and peripheralcomponents on the peripheral bus 118. In some alternative architectures,the functionality of the north bridge can be incorporated into theprocessor instead of using a separate north bridge chip.

In some embodiments, system 100 can include an accelerator card 122attached to the peripheral bus 118. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing. For example, an accelerator can be used for adaptivedata restructuring or to evaluate algebraic expressions used in extendedset processing.

Software and data are stored in external storage 124 and can be loadedinto RAM 110 and/or cache 104 for use by the processor. The system 100includes an operating system for managing system resources; non-limitingexamples of operating systems include: Linux, Windows™, MacOS™,BlackBerry OS™, iOS™, and other functionally-equivalent operatingsystems, as well as application software running on top of the operatingsystem for managing data storage and optimization in accordance withexample embodiments of the present invention.

In this example, system 100 also includes network interface cards (NICs)120 and 121 connected to the peripheral bus for providing networkinterfaces to external storage, such as Network Attached Storage (NAS)and other computer systems that can be used for distributed parallelprocessing.

FIG. 17 is a diagram showing a network 200 with a plurality of computersystems 202 a, and 202 b, a plurality of cell phones and personal dataassistants 202 c, and Network Attached Storage (NAS) 204 a, and 204 b.In example embodiments, systems 202 a, 202 b, and 202 c can manage datastorage and optimize data access for data stored in Network AttachedStorage (NAS) 204 a and 204 b. A mathematical model can be used for thedata and be evaluated using distributed parallel processing acrosscomputer systems 202 a, and 202 b, and cell phone and personal dataassistant systems 202 c. Computer systems 202 a, and 202 b, and cellphone and personal data assistant systems 202 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 204 a and 204 b. FIG. 17 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various embodiments of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some example embodiments, processors can maintain separate memoryspaces and transmit data through network interfaces, back plane or otherconnectors for parallel processing by other processors. In otherembodiments, some or all of the processors can use a shared virtualaddress memory space.

FIG. 18 is a block diagram of a multiprocessor computer system using ashared virtual address memory space in accordance with an exampleembodiment. The system includes a plurality of processors 302 a-f thatcan access a shared memory subsystem 304. The system incorporates aplurality of programmable hardware memory algorithm processors (MAPs)306 a-f in the memory subsystem 304. Each MAP 306 a-f can comprise amemory 308 a-f and one or more field programmable gate arrays (FPGAs)310 a-f. The MAP provides a configurable functional unit and particularalgorithms or portions of algorithms can be provided to the FPGAs 310a-f for processing in close coordination with a respective processor.For example, the MAPs can be used to evaluate algebraic expressionsregarding the data model and to perform adaptive data restructuring inexample embodiments. In this example, each MAP is globally accessible byall of the processors for these purposes. In one configuration, each MAPcan use Direct Memory Access (DMA) to access an associated memory 308a-f, allowing it to execute tasks independently of, and asynchronouslyfrom, the respective microprocessor 302 a-f. In this configuration, aMAP can feed results directly to another MAP for pipelining and parallelexecution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleembodiments, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some embodiments,all or part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example embodiments, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example embodiments, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other embodiments, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 18, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 122 illustrated in FIG. 16.

EXAMPLE 1—DETERMINATION OF STRUCTURAL PARAMETERS IN DIHYDROFOLATEREDUCTASE (DHFR) MUTANTS

Glassware and Sonicated Lipid Preparation:

Clean all glassware with Piranha wash (20 minutes) prior to starting.Use caution—Piranha wash is highly exothermic and prone to explosion,especially when in contact with organics. Prepare a solution inheat-safe glassware such as Pyrex in a fume hood by measuring out H₂O₂first, then adding acetic acid. Rinse vacuum bottles with Chloroform(CHCl₃). Determine desired molar ratio of DOPC lipid to DGS NTA-Ni whiletaking care to avoid exposure to air as much as possible. Place vacuumbottle with lipid mix onto a Rotovap evaporator. Evaporate until dry(about 30 seconds) and then blow N₂ gas over the evaporated preparationfor 10 min to remove residual CHCl₃. Resuspend the lipid mixture in 2 mLof diH₂O. Vortex vigorously until a cloudy suspension forms (about 5minutes). Transfer the suspension to a 4 mL polystyrene test tube.Sonicate the lipid mixture on ice until the solution clears. This shouldrequire about 60 to 90 seconds with the sonicator set to 25% power.

Transfer the sonicated lipid solution into microcentrifuge tubes andcentrifuge at 17,000×G for 30 minutes at 4° C. Transfer the supernatantinto clean microcentrifuge tubes and store the finished lipid preps at4° C. which are stable for about 1 month.

Slide Preparation and Protein Loading:

Immediately before applying DOPC/DGS NTA (Ni) lipids, clean microscopeslides with Piranha wash for 20 minutes. Rinse 3× with diH₂O in a slidestaining vessel. Dry slides with compressed Nitrogen. Assemble SHG wellsby attaching adhesive gaskets to Piranha-cleaned slides (i.e., 16 wellsper slide containing 10-20 μl volume). Use an assembly jig to aligngaskets, carefully lay slide into jig and press firmly. Dilute DOPC/DGSNTA (Ni) lipid prep 1:1 with PBS or TBS buffers. 100 mM NaCl is requiredto reduce hydrostatic charge of the glass slide and enable the supportedlipid bilayer (SLB) to form. Pipet 10-20 μL of diluted DOPC/DGS NTA (Ni)lipid into the wells of the slide and incubate for 5 minutes at roomtemperature. Wash the wells by submersing the slide in buffer bath (PBSor TBS) and agitating with a 200 μL pipettor taking care not tointroduce air into the wells at any time. Exchange the entire volume ofbuffer in the bath with fresh buffer and repeat the washing step 2 moretimes. Add a 1:1 volume of 100 mM NiCl₂ solution to all wells andincubate for 10 minutes at room temperature. Wash the wells bysubmersing the slide in buffer bath (PBS or TBS) and agitating with a200 μL pipettor. Exchange the entire volume of buffer in bath with freshbuffer and repeat the washing step 2 more times. If necessary, exchangethe buffer in the wells to an appropriate protein loading buffer andload the target protein of interest onto the wells. Incubate for 30 to90 minutes at room temperature followed by a thorough rinse of the wellswith assay buffer before starting experiments.

Small unilamellar vesicles (SUVs) are prepared by sonication asdescribed above and applied over Piranha-washed Fisher slides to makethe SLB surface. NiCl₂ was added for 10 minutes and wells were washed inlabeling buffer.

Labeled protein is loaded onto the SLB surface prepared as describeabove at 3 μM (micromolar) for 45 minutes, followed by washing. Ifimidazole or EDTA is added or the protein is incubated with the SLBsurface in the presence of one or both, the SHG signal drops to thebaseline level indicating that attachment to the surface occursspecifically via the protein's His-tag.

A mutant of the Escherichia coli protein dihydrofolate reductase (DHFR)with either an N-terminal or C-terminal 8× His tag was created usingmethods known in the art (Rajagopalan, P., et al. (2002), “Interactionof Dihydrofolate Reductase with Methotrexate: Ensemble andSingle-Molecule Kinetics”, Proc. Nat. Acad. of Sci. (USA)99(21):13481-6; Goodey, N. (2008), “Allosteric Regulation and CatalysisEmerge via a Common Route”, Nat Chem. Biol. 4(8):474-82; Antikainen, N.(2005), “Conformation Coupled Enzyme Catalysis: A Single-Molecule andTransient Kinetics Investigation of Dihydrofolate Reductase”,Biochemistry 44(51):16835-43). In the first case, a cysteine minimizedmutant was made in which both native cysteines were removed (C85A andC152A). Then two single, different residues (e.g., M16C, N23C, Q65C,V136C, D142C, A19C, etc.) were mutated to cysteine, i.e. two single-sitemutant constructs were created in this cysteine minimized background:mutant 1 and mutant 2. To select the site for the mutation, variousresidues on the surface of the protein were mutated to cysteine andtested for an ability to be labeled by an SHG probe. In the second case,the wild-type protein was labeled and attachment of the probe to C152was confirmed by mass spectrometry. Wild type or recombinant protein waspurified into 25 mM Tris pH 7.2, 150 mM NaCl. The proteins were labeledusing a maleimide dye following the manufacturer's instructions, e.g.protein was incubated in 25 mM Tris pH 7.2, 150 mM NaCl, 1 mM TCEP and10% glycerol at approximately 50 uM with a 20:1 dye:protein labelingratio (final DMSO concentration was 5%). The protein was stirredovernight at 4° C., and then gel-purified into 25 mM Tris-HCl pH 7.2,150 mM NaCl, 1 mM TCEP. Labeled and purified protein was then tetheredto the SLB surface via the His-tag.

Polarization-dependent measurements of the SHG signal were used todetermine the independent, non-vanishing components of χ⁽²⁾ for the twodifferent mutants using methods known to those skilled in the art. Forexample, in the simplest optical geometry, and assuming azimuthalisotropy and a single dominant component of the hyperpolarizabilitytensor, one determines two independent non-vanishing components of χ⁽²⁾(χ_(zzz) and χ_(xzx) or χ_(zxx)). These in turn were used to bestdetermine the orientational distributions related to the θ's for each ofthe mutants (i.e., θ₁ and θ₂).

For example, for V136C, Q65C, and N23C labeled at the single-sitecysteines tethered to the lipid bilayer via a His-tag at the C-terminus,the mean angle of the dye hyperpolarizability relative to the surfacenormal (z-axis), assuming a Delta function in orientation (single angleand no width in the orientational distribution), was as follows:

V136C 35.3° Q65C 47.5° N23C 38.5°

FIGS. 13A-B shows the I_(zzz) measurements for various SHG-labeledsingle-site cysteine mutants tagged and tethered by the His tag ateither the N-terminus or the C-terminus. As was immediately obvious, theabsolute signal magnitudes of the labeled proteins (the baselinesignals) varied from label site to label site for N-terminal andC-terminal tagged and tethered proteins (FIG. 13A and FIG. 13B,respectively).

Addition of ligand caused changes in the SHG baseline signal levels (SHG% changes), either positive or negative, which result from reorientationof the labels across the protein ensemble, in other words, from changesin the label orientational distribution as a result of ligand binding.FIGS. 14A and 14B show the results of an experiment in which 1 uM(micromolar) TMP ligand, which is well known to bind DHFR to thoseskilled in the art, produced differential changes in baseline signal(I_(zzz)). As can be seen in FIGS. 14A and 14B, the direction andmagnitude of the SHG signal changes varied from site to site both forcommon tagging and tethering (e.g., N-terminal or C-terminal tagging)and across the results for N- and C-terminal tagged mutants. Forexample, as seen in FIG. 14A, addition of TMP causes a roughly 70%decrease in baseline signal for the protein mutant that is attached andoriented via it's His tag at the N-terminus, but an approximately 90%decrease for the mutant that is attached and oriented via a His tag atthe C-terminus.

Similar measurements can be repeated for I_(zxx), and a ratio of the twopolarization-dependent intensities can be used to determine the meanangle of the label at each cysteine site as a function of proteinconstruct (e.g., N- or C-terminus tag) assuming a narrow orientationaldistribution.

EXAMPLE 2 (PROPHETIC)

The experiment of Example 1 may be extended to include 3 or moredifferent His-tag lengths which result in different orientationaldistributions to be observed. This increases the number of independentpolarization measurements to 48 and increases the accuracy of theangular measurements and the protein structural models.

EXAMPLE 3 (PROPHETIC)

The experiment of Example 2 may be extended to include 4 differentbuffers of varying salt concentration (e.g. 0, 50 mM, 200 mM and 300 mMNaCl) which produces different orientational distributions. Thisincreases the number of independent polarization measurements to 384 andincreases the accuracy of the angular measurements and the proteinstructural models.

EXAMPLE 4 (PROPHETIC)

The experiment of Example 3 may be extended to include differentconcentrations of a molecule added to the buffer (i.e. an additive)(e.g. PEG400 (10 uM, 20 uM, 40 uM and 80 uM micromolar PEG400)) whichassociates with the interfacial region and produces differentorientational distributions. This increases the number of independentorientational distributions and polarization measurements and thusincreases the accuracy of the angular measurements and the proteinstructural models.

EXAMPLE 5 (PROPHETIC)

The experiment of Example 4 may be extended to include a transverseE-field applied across the supported lipid bilayer according to methodsknown to those skilled in the art. Four different electric fieldstrengths (zero, low, medium, and high) are applied. Metallic strips arepatterned across a bilayer region in a 384-well glass-bottom plate well(˜3 mm diameter), and a voltage ranging from 0.1-100 V is applied tothese strips corresponding to field strengths of ˜1 V/cm-1E⁴ V/cm. Theapplied electric field alters the orientational distribution of theprotein population via electrophoretic and electroosmotic mechanisms asis known to those skilled in the art; different magnitudes appliedelectric field result in different orientational populations of theprotein and allow for independent polarization measurements. Thisincreases the accuracy of the angular measurements and the proteinstructural models.

EXAMPLE 6

The experiment of Example 5 may be extended to include 10 differentmutants of DHFR, each labeled at a different single cysteine site. Thisincreases the number of independent polarization measurements and theaccuracy of the angular measurements and the protein structural models.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for determining protein structure insolution, the method comprising: (a) tethering protein molecules to asurface under three or more different sets of experimental conditionsknown to produce different orientational distributions of the tetheredprotein molecules, wherein the protein molecules are labeled at one ormore known positions with one or more nonlinear-active labels for eachexperimental condition, and wherein the three or more different sets ofexperimental conditions comprise two or more of: (i) tethering theprotein molecules using a His-tag attached to the N-terminus, (ii)tethering the protein molecules using a His-tag attached to theC-terminus, and (iii) tethering the protein molecules to at least twosurfaces of different composition; (b) illuminating the tethered proteinmolecules of step (a) with excitation light of at least one fundamentalfrequency and a first polarization, wherein the excitation light isprovided by at least one light source; (c) detecting a first intensityof light generated by the one or more nonlinear-active labels as aresult of the illumination in step (b) for the three or more differentsets of experimental conditions; (d) illuminating the tethered proteinmolecules of step (a) with excitation light of the at least onefundamental frequency and a second polarization; (e) detecting a secondintensity of light generated by the one or more nonlinear-active labelsas a result of the illumination in step (d) for the three or moredifferent sets of experimental conditions; (f) calculating intensityratios for the light detected at the first and second polarizations foreach set of experimental conditions to determine a relative orientationof the one or more nonlinear-active labels in the tethered proteinmolecules for each set of experimental conditions; and (g) globallyfitting data for the relative orientation of the one or morenonlinear-active labels under each set of experimental conditions to astructural model of the protein molecule, wherein the structural modelis based on known positions of the one or more nonlinear-active labelswithin the protein molecule.
 2. The method of claim 1, furthercomprising repeating steps (a) through (f) for at least two differentnonlinear-active label-protein conjugates, wherein the nonlinear-activelabels are attached to at least two different sites on the proteinmolecule.
 3. The method of claim 2, wherein the at least two differentnonlinear-active label-protein conjugates each comprise a single-sitecysteine.
 4. The method of claim 1, wherein the nonlinear-active labelsare nonlinear-active unnatural amino acids.
 5. The method of claim 1,wherein the first and the at least second physical properties of lightpossess the same polarization but are of different magnitudes orintensities.
 6. The method of claim 1, wherein the first and at theleast second physical properties of light possess differentpolarizations.
 7. The method of claim 4, wherein the nonlinear-activeunnatural amino acid is Aladan or a derivative of naphthalene.
 8. Themethod of claim 1, further comprising incorporating x-raycrystallographic data for the protein into the structural model of theprotein molecule.
 9. The method of claim 1, wherein one of the three ormore different sets of experimental conditions comprises applying afirst electric field of a first electric field strength to the tetheredprotein molecules, and another one the three or more different sets ofexperimental conditions comprises applying a second electric field of asecond electric field strength to the tethered protein molecules. 10.The method of claim 9, wherein the first electric field and the secondelectric field are selected from the group consisting of direct current(DC) fields, alternating current (AC) fields, and any combinationthereof.
 11. The method of claim 9, wherein the first electric field andthe second electric field are applied using an array of electrodesfabricated on the surface.
 12. The method of claim 11, wherein the arrayof electrodes is a circular array as illustrated in FIG.
 9. 13. Themethod of claim 1, wherein one of the three or more different sets ofexperimental conditions comprises tethering the protein molecules usinga first His-tag selected from the group consisting of 2×His, 4×His,6×His, 8×His, 10×His, 12×His, and 14×His, and another one of the threeor more different sets of experimental conditions comprises tetheringthe protein molecules using a second His-tag that differs in length fromthe first His-tag.
 14. The method of claim 1, wherein one of the threeor more different sets of experimental conditions comprises tetheringthe protein molecules using a first assay buffer, and another one of thethree or more different sets of experimental conditions comprisestethering the protein molecules using a second assay buffer that differsfrom the first assay buffer.
 15. The method of claim 14, wherein thedifference between the first assay buffer and the second assay buffer isselected from the group consisting of ionic strength, pH, detergentconcentration, calcium ion (Ca²⁺) concentration, magnesium ion (Mg²⁺)concentration, polyethylene glycol concentration, and any combinationthereof.
 16. The method of claim 1, wherein the difference between oneof the three or more different sets of experimental conditions andanother one of the three or more different sets of experimentalconditions comprises contacting the tethered protein molecules with atleast a first ligand that is known to bind to and induce conformationalchange in the protein molecules.
 17. The method of claim 1, wherein theone or more nonlinear-active labels located at the one or more knownpositions are the same.
 18. The method of 1, wherein the one or morenonlinear-active labels located at the one or more known positions aredifferent.
 19. The method of claim 1, wherein the one or morenonlinear-active labels are selected from the group consisting of secondharmonic (SH)-active labels, sum frequency (SF)-active labels, anddifference frequency (DF)-active labels.
 20. The method of claim 1,wherein the detecting in steps (c) and (e) comprise adjusting thepolarization of the light generated by the one or more nonlinear-activelabels that reaches a detector.
 21. The method of claim 1, wherein theone or more nonlinear-active labels are selected from the groupconsisting of pyridyloxazole (PyMPO), PyMPO succinimidyl ester, andPyMPO maleimide.